# OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem

Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, Federico Podico, Elahe Tavakol, François Parcy, Andrea Gallavotti, Vittoria Brambilla, et al.

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## To cite this version:

Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, et al.. OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem. New Phytologist, 2021, 229 (1), pp.429-443. 10.1111/nph.16834. hal-02953028

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1 OsFD4 promotes the rice floral transition via Florigen Activation Complex formation in the
2 shoot apical meristem

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4 Martina Cerise¹,⁶, Francesca Giaume¹, Mary Galli³, Bahman Khahani⁴, Jérémy Lucas⁵, Federico
5 Podico¹, Elahe Tavakol⁴, François Parcy⁵, Andrea Gallavotti³, Vittoria Brambilla² and Fabio
6 Fornara¹,*

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8 ¹Department of Biosciences, University of Milan, Milan, 20123 Italy
9 ²Department of Agricultural and Environmental Sciences, University of Milan, Milan, 20123 Italy

10 ³Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey, NJ 08854,
11 United States
12 ⁴Department of Crop Production and Plant Breeding, College of Agriculture, Shiraz University,
13 Shiraz, 71441-65186, Iran

14 ⁵University Grenoble Alpes, CNRS, CEA, INRAE, IRIG-DBSCI-LPCV, 17 avenue des martyrs, F-
15 38054, Grenoble, France

16 ⁶Max Planck Institute for Plant Breeding Research, Department of Developmental Biology,
17 Cologne, D-50829, Germany

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19 *Correspondence: Fabio Fornara (fabio.fornara@unimi.it), Tel. +390250314817

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21 Total word count: 6515
22 Introduction word count: 1163
23 Materials and Methods word count: 852
24 Results word count: 2628
25 Discussion word count: 1866
26 Number of figures: 6

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28 Heading title: OsFD4 promotes flowering in rice
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30 Associated twitter accounts: @fornaralab, @vittoriabr, @Francois_Parcy

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35 ORCID DETAILS

36 Martina Cerise, https://orcid.org/0000-0002-9654-252X

37 Francesca Giaume, https://orcid.org/0000-0002-6074-6950

38 Bahman Khahani, https://orcid.org/0000-0003-4499-2825

39 Elahe Tavakol, https://orcid.org/0000-0001-9033-7237

40 François Parcy, https://orcid.org/0000-0003-2191-500X

41 Andrea Gallavotti, https://orcid.org/0000-0002-1901-2971

42 Vittoria Brambilla, https://orcid.org/0000-0003-0673-6898

43 Fabio Fornara, https://orcid.org/0000-0002-1050-0477

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# SUMMARY

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* In rice, the florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), OsFD-like bZIP transcription factors, and Gf14 proteins assemble into Florigen Activation/Repressor Complexes (FACs/FRCs), which regulate transition to flowering in leaves and apical meristem. Only OsFD1 has been described as part of complexes promoting flowering at the meristem, and little is known about the role of other bZIP transcription factors, the combinatorial complexity of FAC formation, and their DNA binding properties.

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* Here, we used mutant analysis, protein-protein interaction assays and DAP-sequencing coupled to *in silico* prediction of binding syntaxes, to study several bZIP proteins that assemble into FACs or FRCs.

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* We identified OsFD4 as component of a FAC promoting flowering at the shoot apical meristem, downstream of *OsFD1*. The *osfd4* mutants are late flowering and delay expression of genes promoting inflorescence development. Protein-protein interactions indicate an extensive network of contacts between several bZIPs and Gf14 proteins. Finally, we identified genomic regions bound by bZIPs with promotive and repressive effects on flowering.

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* We conclude that distinct bZIPs orchestrate floral induction at the meristem and that FAC formation is largely combinatorial. While binding to the same consensus motif, their DNA binding syntax is different, suggesting discriminatory functions.

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Key words: bZIP, florigen activation complex, photoperiodic flowering, rice, shoot apical meristem.

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103 INTRODUCTION

104 When external and internal conditions are favourable, plants switch their life cycle from the
105 vegetative to the reproductive phase. This process is called floral transition and is determined by
106 events taking place in both leaves and shoot apical meristem (SAM). In rice (Oryza sativa), transition
107 to the reproductive phase is controlled by changes in the photoperiod and is accelerated when day
108 length falls under a critical threshold. Because of this response, rice is classified as short day (SD)
109 plant. Yet, it can flower also under non-inductive long day (LD) conditions, after a prolonged
110 vegetative phase. As such, its photoperiodic response is facultative.

111 The molecular events that guide the floral transition start in the leaves when transcription of Heading
112 date 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) takes place in response to inductive
113 photoperiods. Hd3a and RFT1 are homologs of Arabidopsis FLOWERING LOCUS T (FT) and
114 members of the phosphatidyl ethanolamine-binding protein (PEBP) family (Kojima et al., 2002;
115 Komiya et al., 2008, 2009). Similarly to FT, they are transcribed in companion cells of leaves;
116 subsequently, their cognate proteins can move through the phloem to reach the SAM, where they
117 promote expression of genes necessary to activate the inflorescence development program (Tamaki
118 et al., 2007; Komiya et al., 2009). Analysis of RNA interference lines and CRISPR mutants
119 demonstrated that a delay in flowering occurs upon downregulation of Hd3a or RFT1 (Komiya et al.,
120 2008, Liu et al., 2019). However, the phenotypic effects of reduced expression of Hd3a or RFT1
121 depend on the photoperiod. Under SD, Hd3a RNAi lines delay flowering and double RFT1-Hd3a
122 RNAi plants completely block it, indicating that both florigens can redundantly promote the floral
123 transition (Komiya et al., 2008; Tamaki et al., 2015). Conversely, RFT1 RNAi lines delay flowering
124 under LD only, indicating that RFT1 can promote flowering also under non-inductive photoperiods
125 (Komiya et al., 2009). This difference correlates with the expression of Hd3a and RFT1 in leaves:
126 when plants grow under SD conditions, both florigens are transcribed, whereas growth under non-
127 inductive LD conditions promotes RFT1 expression only (Komiya et al., 2008, 2009). Differential
128 sensitivity of Hd3a and RFT1 transcription to day length is partly explained by distinct promoter
129 architectures (Komiya et al., 2008, 2009).

130 Several positive and negative regulators control expression of the florigens in leaves. Among them,
131 the zinc finger transcription factor Heading date 1 (Hd1) , homolog of Arabidopsis CONSTANS,
132 promotes expression of Hd3a and RFT1 under SD, while it represses it under LD (Yano et al., 2000;
133 Hayama et al., 2003). Hd1 functions as transcriptional regulator through physical interaction with
134 Grain yield, plant height and heading date 8 (Ghd8), Ghd7 and OsNF-YC7. Ghd8 and OsNF-YC7
135 are the B and C subunits of a heterotrimeric NF-Y complex, respectively, that assemble with Hd1,
136 bind the promoter of Hd3a and repress its transcription (Goretti et al., 2017; Zhu et al., 2017; Du et
al., 2017). *Ghd7* encodes for a CCT domain protein that represses flowering (Xue *et al.*, 2008; Nemoto *et al.*, 2016).

The B-type response regulator *Early Heading date 1* (*Ehd1*) is a major flowering promotor that shares no homology with other genes of *Arabidopsis thaliana* (Doi *et al.*, 2004). *Ehd1* is expressed at higher levels under SD conditions and preferentially promotes *Hd3a* transcription (Zhao *et al.*, 2015). In agreement with its role as a flowering activator, *ehd1* mutants show a late flowering phenotype compared to wild type, whereas *Ehd1* overexpression triggers early flowering under both SD and LD conditions (Doi *et al.*, 2004; Itoh *et al.*, 2010; Cho *et al.*, 2016). Hd1 can repress *Ehd1* mRNA expression under LD, by directly binding to its promoter (Nemoto *et al.*, 2016). These genetic interactions are at the core of the photoperiodic flowering pathway in rice, which exhibits a different architecture compared to the one of Arabidopsis (Andrés & Coupland, 2012).

When florigenic proteins are produced, they influence downstream gene expression by assembling into Florigen Activation or Repressor Complexes (FACs/FRCs), higher-order complexes containing PEBP molecules and FD-like bZIP transcription factors (Tsuji *et al.*, 2013; Brambilla *et al.*, 2017; Kaneko-Suzuki *et al.*, 2018). The latter allow the complex to bind DNA at consensus sites characterized by the core sequence ACGT (Izawa *et al.*, 1993). Interactions between florigens and bZIPs can occur either directly or indirectly via bridging proteins called Gf14s, members of the 14-3-3 protein family (Taoka *et al.*, 2011). In leaves, FACs/FRCs regulate expression of the florigens, generating positive and negative feedback loops controlling *Ehd1* expression (Brambilla *et al.*, 2017). More specifically, FRCs composed by the florigens and the bZIP transcription factor Hd3a BINDING REPRESSOR FACTOR 1 (HBF1) delay the floral transition, as rice loss-of-function *HBF1* mutants flower earlier compared to the wild type. HBF1 can bind the promoter of *Ehd1* to reduce its expression in leaves, thus generating a negative feedback loop on *Hd3a* and *RFT1* production, that likely modulates the amount of florigens being produced (Brambilla *et al.*, 2017).

Another bZIP transcription factor, *OsFD1*, encodes an interactor of the florigens and is part of a flowering-promoting FAC (Taoka *et al.*, 2011). *OsFD1* is expressed in both leaves and SAM and interacts with Hd3a and RFT1 only indirectly via Gf14 proteins. In leaves, OsFD1 promotes the expression of *Ehd1* generating a positive feedback loop on florigen production, whereas in the SAM, OsFD1-containing FACs promote transcription of *OsMADS14* and *OsMADS15*, two key activators of flower development in rice (Taoka *et al.*, 2011; Kobayashi *et al.*, 2012; Zhao *et al.*, 2015; Wu *et al.*, 2017). Thus, the florigens not only connect leaves and SAM to convey seasonal information to the apex, but their movement throughout the plant allows formation of distinct florigen-containing complexes in different tissues and with diversified functions.
In the SAM, OsFD1 can further switch from activating to repressing flowering by forming FRCs. In 170 FRCs, OsFD1 interacts with four different CENTRORADIALIS-like proteins, RICE 171 CENTRORADIALIS 1 (RCN1), RCN2, RCN3 and RCN4 which are PEBP and members of the TFL1-172 like subclade (Kaneko-Suzuki et al., 2018). They are expressed in the rice vascular tissue and, 173 similarly to Hd3a and RFT1, can move through the phloem and reach the SAM where they bind 174 OsFD1 via Gf14s, to regulate production of secondary branches of the panicle (Kaneko-Suzuki et al., 175 2018). 176

Despite its central position in molecular complexes controlling flowering, *osfd1* RNA interference 177 plants showed almost no flowering delay compared to wild type plants (Taoka *et al*., 2011). Its 178 interaction in both FACs and FRCs with opposite effects on flowering could partially explain such 179 phenotype. However, it cannot be excluded that additional bZIP transcription factors could operate 180 in parallel or redundantly with OsFD1. 181

Here, we functionally characterize *OsFD4*, an FD-like bZIP transcription factor promoting the floral 182 transition under both inductive and non-inductive photoperiodic conditions. We determine the extent 183 of OsFD4 interactions in FACs and its genetic relationship with OsFD1. Using Positional Weight 184 Matrices (PWM) gathered from DNA Affinity Purification sequencing (DAP-seq), we interrogate the 185 binding of OsFD1, OsFD4 and HBF1 to promoters of target genes, starting to define their distinct 186 modes of DNA binding. 187

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## 189 MATERIALS AND METHODS

190 Plant growth conditions, RNA preparation and quantification of gene expression

The Nipponbare and Dongjin ecotypes (*Oryza sativa* L.) were used in this study. Plants were grown 191 in phytotrons under long-day conditions (14,5h light/9,5h dark) or short-day conditions (10h light/14h 192 dark). For quantification of gene expression using qRT-PCR, SAM samples were collected at 193 *Zeitgeber* (ZT) 0 in time courses of several days. Three biological replicates were performed for 194 *osfd4-1* time-courses. Two biological replicates were sampled for *osfd1-1* time courses. SAMs were 195 manually dissected under a stereomicroscope and the samples included the meristem proper as well 196 as some small meristematic leaves. For leaf samples, the distal part of mature leaves was used. RNA 197 was extracted with *TRIzol®* (Termofisher Scientific) following manufacturer's instructions. The 198 cDNA was retrotranscribed with Im-Prom-II RT (Promega) using a polyT primer and 1 µg of total 199 RNA as starting template. Quantification of transcripts was performed with the primers listed in Table 200 S1 using a RealPlex² thermocycler. The Maxima SYBR qPCR master mix (Termofisher Scientific) 201 was used in qRT-PCR experiments. Three technical replicates were performed for each qRT-PCR. 202 203
204 Protein-protein interaction assays

205 Yeast two hybrid was performed cloning the coding sequences into the vectors pGADT7 and
206 pGBKT7 (Clontech) and transformed into AH109 and Y187 yeast strains, respectively. Interactions
207 were tested by mating and growth on selective drop out media without the aminoacid -L-W-H-A
208 according to Brambilla et al., 2017.

209 BiFC experiments were performed using the vectors pBAT TL-B sYFP-N and pBAT TL-B sYFP-C.
210 Clones were transformed in tobacco epidermal cells by Agro-infiltration. Infiltrated leaves were
211 observed at the confocal 24 h after infiltration. Each interaction was tested in three different replicates.
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213 CRISPR-Cas9 cloning and Rice transformation

214 The CRISPR-Cas9 vectors previously described by Miao et al., 2013 were used. We designed one
215 single-guide RNA for OsFD1, OsFD4 and OsFD3 (Table S1). We obtained edited *osfd4*, *osfd3* and
216 *osfd1* mutants starting from Nipponbare calli produced according to the protocol of Sahoo et al., 2011.

217 Rice calli were transformed using EHA105 *Agrobacterium tumefaciens* strain. Transformed calli
218 were selected on two selection media containing 50 mg/L and 100 mg/L of hygromycin, respectively.

219 After regeneration, we genotyped transformant plants via by sequencing PCR products amplified
220 across the single-guide RNA targeted region.

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222 DAP seq assay

223 pENTR clones containing the *OsBZIP42*, *OsBZIP69*, and *OsBZIP77* ORFs were recombined into the
224 pIX-HALO vector and processed according to Bartlett et al., 2017. We used 1 ug of pIX-HALO-TF
225 plasmid DNA for protein expression in the TNT rabbit reticulocyte expression system (Promega).

226 For DNA binding reactions, 500 ng of adapter-ligated library prepared from genomic DNA extracted
227 from Nipponbare leaves was used. Libraries were sequenced on a NextSeq500 with 75bp single end
228 reads. A GST-HALO negative control sample was also processed in parallel and used for background
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231 DAP-seq analysis

232 Raw reads were trimmed using Trimmomatic (Bolger et al., 2014) with the following parameters:
233 ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
234 MINLEN:50. Trimmed reads were mapped to the *Osativa* v7 reference genome (release 40;
235 https://plants.ensembl.org/Oryza_sativa/) using Bowtie2 v2.2.8 (Langmead & Salzberg, 2012) with
236 default parameters. Mapped reads were filtered for reads aligning only to a single location in the
237 genome. Peaks were called using the GEM peak caller (Guo et al., 2012) as described in Galli et al.,
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2018. PAVIS (Huang et al. 2013) was used to identify putative target genes. Target genes were assigned based on the gene located closest to each peak and were restricted to those peaks located 5kb upstream of the TSS, 3kb downstream of the TES, or located within the gene. The 2000 interval relative to the TSS was surveyed for detecting the position of peaks and distribution of them using a bin size of 25 bp.

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## Comparison between targets of AtFD, OsFD1, OsFD4 and OsHBF1

In order to compare the FD-bound regions in *Arabidopsis* and rice, we retrieved ChIP-seq data for FD from Collani et al. (2019). We next identified putative rice orthologs using the EnsemblPlants (https://plants.ensembl.org/index.html) database (Table S2). The overlap between corresponding genes bound by AtFD, OsFD1, OsFD4 and HBF1 was illustrated using a Venn diagram (Oliveros, 2007).

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## Motif enrichment analysis and spacing/syntax analysis

Peaks identified by the GEM (Guo et al., 2012) peak caller were used to compute motif enrichment, using the MEME-suite (default arguments). Generated motifs were evaluated using ROC analysis to estimate their prediction power, i.e. their capability to differentiate between bound and not bound regions. For this, a negative set of non-bound regions with similar features (same length, same %GC, same genomic origin [exonic, promoter,...] as the bound sequences) was generated as previously described (Stigliani et al., 2019). Detection of preferred spacings between motifs was done using the called peaks and the MEME-generated position weight matrix (PWM). Predicted individual binding sites were detected for a given threshold, for sets of both bound and unbound regions. Spacing between predicted binding sites were computed for both sets. Enrichment was calculated by comparing bound and not bound sets of regions. The PWM threshold values were empirically chosen: we used a range of threshold values to ensure robustness of the result. Detailed methods are available in Stigliani et al., 2019.

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# RESULTS

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## OsFD4 promotes flowering in rice

In leaves, HBF1- and OsFD1-containing complexes control production of the florigens by regulating *Ehd1* expression (Brambilla et al., 2017). In the SAM, the OsFD1-containing FAC is known to promote the floral transition by directly inducing expression of *OsMADS14* and *OsMADS15* (Taoka et al., 2011). Yet, *OsFD1* RNA interference plants showed only a marginally significant flowering delay compared to the wild type, in sharp contrast to the phenotype shown by *Hd3a*-RNAi, *RFT1*-

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272 RNAi and Hd3a/RFT1 double RNAi plants (Komiya et al., 2008). This suggests that other bZIP
273 transcription factors might share similar functions and compensate for reduced OsFD1 expression.
274 We previously explored the bZIP family, searching for members having positive or negative effects
275 on the floral transition (Brambilla et al., 2017). Here, we describe the functional characterization of
276 bZIP69/OsFD4 (LOC_Os08g43600).

277 After searching the PFG rice T-DNA collection we identified line 2D_41663 in the Dongjin variety
278 that harboured a T-DNA insertion in the 3'-UTR of OsFD4 (Fig. S1). The mutant line was renamed
279 osfd4-1. We could not detect OsFD4 mRNA expression in osfd4-1 (Fig. S1). Heading dates of the
280 mutant plants were assessed under both SD and LD, alongside Dongjin wild type controls. Mutant
281 plants flowered late under both conditions (Fig. 1a).

282 To validate the *osfd4-1* mutant phenotype, we generated additional *osfd4* mutant alleles using
283 CRISPR-Cas9 site-directed mutagenesis in the Nipponbare background (Miao *et al.*, 2013). We also
284 generated novel *osfd1* mutants to compare single mutant phenotypes. Finally, since we previously
285 showed that OsFD4 strongly interacts with bZIP24/OsFD3 (Brambilla et al., 2017), we also generated
286 *osfd3* mutants (Fig. S1). Homozygous plants of the T1 generation were grown for two months under
287 non-inductive LD conditions and then shifted to inductive SD to score heading dates. As shown in
288 Fig. 1b and Fig. S1, *osfd4-3* showed delayed flowering compared to the wild type, confirming the
289 *osfd4-1* phenotype. The *osfd1-1* mutant flowered later than both wild type and *osfd4-3*, showing a
290 strong delay (Fig. 1b-d). The difference compared to published *OsFD1*-RNAi mutants could be due
291 to the different conditions used between studies, to the different genetic backgrounds used, or to a
292 limited reduction of *OsFD1* mRNA in RNAi lines. No effect on heading could be observed in *osfd3-1*
293 mutants (Fig. 1b).

294 To investigate the possible functional redundancy between *OsFD4* and *OsFD1*, we crossed *osfd4-1*
295 and *osfd1-2* mutants to generate an *osfd4-1 osfd1-2* double mutant (note that the genetic background
296 is mixed between Nipponbare and Dongjin). Flowering time experiments under inductive conditions
297 demonstrated that no additive effect could be detected in the *osfd4-1 osfd1-2* double mutant relative
298 to *osfd1-2* (Fig. 1d), i.e. the phenotypic effect observed was that of *osfd1-2* single (Fig. 1b-d). These
299 data indicate that *OsFD4* encodes a promotor of flowering in rice and that the effect of *osfd4* and
300 *osfd1* mutants is not additive.

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302 *OsFD4* is expressed in the shoot apical meristem and is regulated by *OsFD1*

303 *OsFD1* is expressed in both leaves and SAM. It promotes flowering, having distinct functions in both
304 tissues during the floral transition (Taoka *et al.*, 2011; Tamaki *et al.*, 2015; Jang *et al.*, 2017;
305 Brambilla *et al.*, 2017). We quantified the expression of *OsFD4* mRNA in leaves and apical
306 meristems, using qRT-PCR. Transcripts of *OsFD4* were detected in apical meristems but not in leaves
307 during the transition to flowering (Fig. 2a,b). Quantifications showed that *OsFD1* and *OsFD3*
308 expression was higher relative to *OsFD4* expression in both tissues.

309 To verify the epistatic interaction between *OsFD1* and *OsFD4*, observed in the *osfd4-1 osfd1-2*
310 double mutant, we analysed *OsFD4* and *OsFD1* mRNA levels in *osfd1-1* and *osfd4-1* mutants,
311 respectively. We observed that *OsFD4* expression was decreased in *osfd1-1* compared to the wild
312 type (Fig. 2c), whereas *OsFD1* expression was unchanged in *osfd4-1* mutants compared to Dj wild
313 type (Fig. 2d). These data indicate that *OsFD1* is an upstream promotor of *OsFD4* expression, in
314 agreement with the phenotype of double *osfd4-1 osfd1-2* mutants (Fig. 1d).

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316 Network of interactions among FAC components

317 The rice florigens interact with bZIP transcription factors either directly or through Gf14 proteins.
318 Interactions are likely dynamic and combinatorial, giving rise to several complexes, some also with
319 repressive functions (Zhao *et al.*, 2015; Jang *et al.*, 2017; Brambilla *et al.*, 2017; Kaneko-Suzuki *et
320 al.*, 2018). Dimers between Gf14 proteins form a W-shaped scaffold that bridges interactions with
321 bZIPs and with the florigens. We found that extensive combinatorial interactions are possible between
322 Gf14s expressed at the apex, giving rise to homo- and heterodimers (Fig. S2, all mating controls are
323 shown in Fig. S3). Interactions between Gf14s and bZIPs occur via the C-terminal SAP-domain,
324 which is common among several bZIPs, including OsFD4 and OsFD3 (Fig. S1). We tested whether
325 OsFD4 could interact with components of FACs. We first tested the binding between OsFD4 and the
326 six Gf14s expressed in the SAM, including Gf14A to Gf14F (Purwestri *et al.*, 2009). All six Gf14
327 proteins interacted with OsFD4 in Yeat-2-Hybrid (Y2H), but the strongest interactions were detected
328 between OsFD4 and Gf14A and Gf14F (Fig. 3a). Weaker yeast growth on -L-W-H-A selective media
329 was observed with other OsFD4-Gf14 combinations. Note that in a previous report we did not detect
330 the OsFD4-Gf14C interaction, using -L-W-H +3AT media (Brambilla *et al.*, 2017), consistent with
331 the findings of Kim *et al.*, 2016. Thus, interactions between OsFD4 and Gf14B, C, D, and E are likely
332 weak. Also, OsFD3 could interact with all Gf14s tested (Fig. S2).

333 We next asked if all Gf14 proteins expressed in the SAM could interact with RFT1 and Hd3a. Both
334 florigens interacted with all the Gf14s tested, suggesting that in the SAM, OsFD4, OsFD3 and OsFD1
335 can potentially form FACs indirectly via all 14-3-3 proteins (Fig. 3b). To confirm Y2H findings, we
336 performed BiFC with OsFD4 and Gf14B and observed reconstituted YFP expression in nuclei
337 indicating a positive interaction (Fig. S2).

338 Since HBF1 and HBF2 can directly contact Hd3a without the bridging function of Gf14s (Brambilla
339 *et al.*, 2017), we tested direct interactions between OsFD4 and Hd3a or RFT1. Using Y2H, we found
that OsFD4 can directly associate with RFT1, but no interaction was detected between OsFD4 and
OsFD3 could interact with both florigens (Fig. 3c). These data are consistent with a
recent independent study, indicating that OsFD4 preferentially binds to one florigen only (Jang *et al.*,
2017). We then used BiFC to further test the OsFD4-RFT1 interaction, as well as to determine its
subcellular localization. We detected fluorescence in nuclei of *Nicotiana benthamiana* leaves
indicating nuclear localization of the heterodimer (Fig. 3d). No fluorescent signals could be detected
for the OsFD4-Hd3a interaction (Fig. 3e and Fig. S2).

### OsFD4 can homodimerize or heterodimerize with OsFD3

bZIP transcription factors must homo- or heterodimerize to bind DNA (Dröge-Laser *et al.*, 2018).
We thus tested combinatorial interactions between bZIPs. We previously showed that OsFD4 and
OsFD3 can form homo- and heterodimers, whereas no dimerization was found with OsFD1
(Brambilla *et al.*, 2017; Fig. 3f,g). Here, we corroborated those results using BiFC, further showing
that OsFD4 and OsFD3 homo- and heterodimers are localized in the nucleus. We did not detect homo-
or heterodimerization of OsFD1 in BiFC assays (Fig. 3h,i).

### OsFD4 regulates expression of AP1/FUL-like genes

The rice genome encodes four different *AP1/FUL*-like genes, *OsMADS14*, *OsMADS15*, *OsMADS18* and *OsMADS20* which control flower development (Wu *et al.*, 2017). When plants are exposed to inductive photoperiods and the meristem undergoes phase change, *OsMADS14*, *OsMADS15* and *OsMADS18* increase their expression, whereas *OsMADS20* is downregulated (Kobayashi *et al.*, 2012; Gómez-Ariza *et al.*, 2019). In the SAM, OsFD1 promotes the expression of *OsMADS14* and *OsMADS15*. This regulation has been shown to be direct at least for *OsMADS15*, as the OsFD1-containing FAC is able to bind to its promoter (Taoka *et al.*, 2011). To assess if an OsFD4-containing FAC could also regulate the expression of *OsMADS* genes we measured transcription of *AP1/FUL*-like genes in apical meristems of *osfd4-1*. We performed time course experiments shifting plants from non-inductive LD conditions to inductive SD conditions and sampled SAMs at 0, 6, 12 and 18 Days After Shifting (DAS). These time points were chosen based on the progression of floral transition in rice which becomes irreversible at 12 SDs, after which a fully committed SAM develops into a branched inflorescence (Kobayashi *et al.*, 2012; Gómez-Ariza *et al.*, 2019). Comparing *OsMADS* gene expression between wild type Dongjin and *osfd4-1* plants, we observed that *OsMADS14* and *OsMADS15* levels could increase in the mutant but at slower rates, whereas expression of *OsMADS18* and *OsMADS20* was not changed compared to the wild type (Fig. 4a-d).
Rice AP1/FUL-like genes work together with OsMADS34/PAP2, a SEPALLATA (SEP)-like gene, to promote the floral transition (Kobayashi *et al.*, 2010, 2012). Expression of OsMADS34/PAP2 increased in both wild type and osfd4-1 but was significantly delayed in the mutant (Fig. 4e). To complete our analysis, we also quantified the expression of AP1/FUL-like genes and of OsMADS34/PAP2 in the osfd1-1 mutant. We observed that their expression had similar dynamics in both osfd1-1 and osfd4-1 (Fig. 4f-j). Taken together, these data suggest that OsFD4-FAC, as well as OsFD1-FAC, act upstream of OsMADS genes to promote the rice floral transition.

## Genome-wide identification of OsFD4, OsFD1 and HBF1 binding sites

In the SAM, changes in the photoperiod regulate the transcription of several genes (Furutani *et al.*, 2006; Kobayashi *et al.*, 2012; Tamaki *et al.*, 2015; Gómez-Ariza *et al.*, 2019). Yet, only a few genes are known to be under control of FACs/FRCs. As a first screen to identify DNA binding sites of bZIP transcription factors and FAC-regulated genes, we performed DNA Affinity Purification sequencing (DAP-seq) using OsFD4, OsFD1 and HBF1 (O'Malley *et al.*, 2016; Bartlett *et al.*, 2017; Galli *et al.*, 2018). Peak calling analysis identified 1107 DAP-peaks for OsFD4, 2059 DAP-peaks for OsFD1, and 28323 DAP-peaks for HBF1 (Fig. 5a). The top enriched motif for OsFD1, OsFD4 and HBF1 contained the core consensus motif CACGT, the known binding site for many bZIP transcription factors known as G-box (Fig. 5a) (O'Malley *et al.*, 2016). At the flanks of this core motif, two nucleotides -GC- were enriched with different frequencies, depending on the bZIP tested. For example, OsFD4 peaks were most enriched for the motif GCCACGT (Fig. 5a).

To identify putative target genes regulated by these binding events, we assigned all DAP-peaks lying within 5000 bp upstream of an ATG and 3000 bp downstream of a STOP codon (including regions within exons and introns) to the closest gene. Based on this analysis, HBF1 peaks were associated with 15,937 putative target genes, and OsFD1 and OsFD4 were associated with 1,717 and 925 putative target genes, respectively (Fig. 5a; Table S3). Most of the binding sites fell in putative promoter regions (Fig. 5b). The highest frequency of peaks (30% for OsFD4, 27% for OsFD1 and 17% for HBF1) was in the 500 bp upstream of the transcription start site (TSS; Fig. 5c), consistent with these sites being highly represented in the core promoter sequences. We next examined the overlap between genes bound by OsFD1, OsFD4 and HBF1 and observed that 698 putative target genes were shared by all tested bZIPs; 220 genes were shared by OsFD4 and HBF1, and 991 were shared by OsFD1 and HBF1. All genes common to OsFD1 and OsFD4 were also common to HBF1, thus no gene was found at the intersection between OsFD1 and OsFD4 datasets only (Fig. 5d and Table S3). Finally, 7, 28 and 14,028 genes were uniquely targeted by OsFD4, OsFD1 or HBF1 (Fig. 5d).
The DAP-seq method uses the entire naked genome to identify transcription factor binding sites. Thus, many bound regions might not be functionally relevant for regulation of gene expression *in vivo* (O'Malley *et al.*, 2016; Bartlett *et al.*, 2017). To focus our analysis on those tissues where OsFD4, OsFD1 and HBF1 are expressed (Fig. 2a), we filtered for genes expressed specifically in the SAM and mature leaves. To this end, we first compared the lists of genes bound by OsFD1 and OsFD4 to those expressed in the shoot apex, based on recently published RNA-seq data (Fig. 5e) (Gómez-Ariza *et al.*, 2019). The lists of genes bound by HBF1 and OsFD1 were compared to the list of genes expressed in mature leaves, again as determined by published RNA-seq data (Galbiati *et al.*, 2016) (Fig. 5f). This analysis identified 240 genes expressed in shoot apices that were putative targets of both OsFD4 and OsFD1, 58 genes that were putative targets of OsFD4, and 320 genes that were putative targets of OsFD1 (Fig. 5e). Of those genes showing expression in mature leaves, 566 were putative targets of both HBF1 and OsFD1, 3691 were putative targets of HBF1, and seven were putative targets of OsFD1 (Fig. 5f).

Finally, we compared putative targets of OsFD1, OsFD4 and HBF1 with targets of AtFD (Collani *et al.*, 2019). To this end, genes identified by ChIP-seq data were retrieved from Collani et al. (2019) and the corresponding putative rice orthologs were assigned based on the EnsemblPlants database (Table S2). The overlap between corresponding genes indicates the existence of targets possibly being part of common regulatory modules, evolutionary conserved (Fig. S4 and Table S4).

### OsMADS62 and OsARF19 are targets of both the OsFD4-FAC and OsFD1-FAC

To determine if binding of OsFD1 and OsFD4 to genomic regions correlated with transcriptional regulation of the neighboring genes, we quantified transcript abundance of selected target genes in *osfd1-1*, *osfd4-1* and wild type. For this analysis, we focused on several transcription factors expressed in the apical meristem and whose promoters contained OsFD1 or OsFD4 binding peaks. These included two genes bound by both *OsFD1* and *OsFD4*, *LOC_Os08g38590* (*OsMADS62*) and *LOC_Os06g48950* (*OsARF19*), three genes bound only by *OsFD1*, *LOC_Os01g14440* (*WRKY*), *LOC_Os01g64360* (*MYB*), and *LOC_Os04g51000* (*RICE FLORICAULA/LEAFY*), and two genes bound by *OsFD4* only, *LOC_Os04g31730* (*B3*) and *LOC_Os07g41580* (*NF-YB*) (Fig. 5g-h and Fig. S5). Expression of both *OsMADS62* and *OsARF19* was reduced in *osfd4-1* and *osfd1-1* compared to the wild type, suggesting that OsFD1 and OsFD4 may be direct activators of both genes (Fig. 5g-h). The other genes tested showed reduced expression in both mutants relative to wild type probably because of the epistatic effect of *OsFD1* to *OsFD4* (Fig. S5). These data suggest that OsFD4 and OsFD1 regulate several common target genes, but their transcriptional effects are not completely overlapping.
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442 OsFD4, OsFD1 and HBF1 show preferential DNA binding configurations

443 OsFD1, OsFD4 and HBF1 bind overlapping but also distinct sets of genes in DAP-Seq experiments, 444 yet they recognize a very similar motif. Because bZIP TFs are known to bind to tandem motifs, we 445 asked if spacing between CACGT consensus motifs found in OsFD1, OsFD4 and HBF1 DAP-seq 446 peaks varied. We used a Position Weight Matrix (PWM) derived from DAP-seq data to locate bZIP 447 binding sites and test whether there are some preferences in terms of binding syntaxes (spacing and 448 configuration between two consensus motifs (Fig. 6a) (Stigliani et al., 2019). We first evaluated the 449 predictive power of the generated PWM which resulted exceptionally high, with ROC values of 450 0.985, 0.971 and 0.9 respectively (Fig. S6). Next, we analyzed the distribution of spacings for each 451 bZIP, quantifying overrepresented configurations against a set of negative regions (unbound), and we 452 found specific profiles for each bZIP.

453 Direct Repeats (DR) 0, Everted Repeats (ER) 1, 5, ER18-20, ER44, Inverted Repeats (IR) 41 454 configurations showed at least a 4-fold overrepresentation among OsFD4 bound regions, with the 455 ER44 configuration being highly overrepresented (Fig. 6b). Regions bound by OsFD1 showed a 456 robust enrichment of at least 3-fold in the DR30, ER4, ER16, ER32-34, ER41-2 and ER46, and IR38 457 configurations (Fig. 6c), with ER16 and ER32-34 as the most overrepresented. Configuration 458 enrichments for HBF1 included DR29, DR40, ER3, ER16-18, ER42 and IR10 with at least 2-fold 459 enrichment compared to the negative set of regions (Fig. 6d). These data identify different spacing 460 configurations between consensus motifs, which are preferred by OsFD4, OsFD1 and HBF1 and 461 likely contribute to the specificity of target recognition.

462

463 DISCUSSION

464 Transcription factors of the bZIP family have important roles during phase transitions in different 465 plant species (Abe et al., 2005; Wigge et al., 2005; Taoka et al., 2011; Tsuji et al., 2013; Park et al., 466 2014; Tylewicz et al., 2015; Brambilla et al., 2017; Teo et al., 2017). However, those controlling 467 flowering time and for which functional studies have been carried out, appear insufficient to account 468 for all aspects of phase change, suggesting a certain level of redundancy, or the existence of additional 469 transcriptional regulators. In rice, the role of OsFD1 as a promoter of flowering has been supported 470 by overexpression studies, but the analysis of RNAi mutants failed to demonstrate phenotypic effects 471 on flowering (Taoka et al., 2011; Jang et al., 2017; Brambilla et al., 2017). Other OsFD-like genes 472 have been investigated previously, however they perform functions distinct from those of OsFD1 473 (Tsuji et al., 2013; Brambilla et al., 2017). Here, to identify additional components of florigen 474 signalling, we reported the characterization of OsFD4 and OsFD3, as well as further insights into
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OsFD1 function based on CRISPR-mediated mutagenesis. While describing such additional FD-like components integrating florigen signalling, it became clear that despite similar modes of action, differences in their expression profiles, specific combinatorial interactions with Gf14 proteins and florigens, and distinct DNA binding syntaxes distinguish different members of the family.

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## The FAC paradigm across Angiosperms

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Formation of a FAC requires that florigenic proteins that have reached the SAM first interact in the cytosol with Gf14 proteins and then enter the nucleus, where phosphorylated bZIP transcription factors contact the Gf14 and contribute a DNA-recognition function. This model of interaction between florigens and bZIP transcription factors was first proposed in Arabidopsis and later supported by studies in rice, where the role of Gf14s as bridges was identified. Subsequently, it has been extended to many other species including dicots and monocots and shown to control a wide range of developmental processes.

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In rice, the functional homolog of Arabidopsis FD is encoded by *OsFD1*. When overexpressed using either a constitutive or phloem-specific promoter, *OsFD1* can accelerate flowering (Jang et al., 2017, Brambilla et al., 2017). However, RNAi lines flower similarly to wild type, possibly *OsFD1* transcripts are not completely absent in these knock-down lines (Taoka *et al.*, 2011). Thus, the functional significance of *OsFD1* as a flowering regulator has remained unclear. Knock-out of *OsFD1* using CRISPR-Cas9 showed that the gene has indeed a prominent effect in promoting flowering. This is likely the result of a more complex molecular function that includes (i) induction of *OsMADS14* and 15 gene expression in the SAM and in leaves, (ii) induction of *Ehd1*, *Hd3a* and *RFT1* mRNA expression in leaves, and (iii) interaction with RCNs in repressor complexes that antagonize FAC-dependent activation of *OsMADS* gene expression. Thus, *OsFD1* and Gf14 proteins are central to both activating and repressive flowering pathways, the switch between them being likely controlled by the relative abundance of FT-like and TFL1-like proteins at the apex.

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Still, while double *Hd3a* and *RFT1* RNAi mutants do not flower for up to 300 days under inductive SDs, single *osfd1* loss-of-function mutants are delayed in flowering but they can flower, an apparent inconsistency justified by functional redundancy between bZIPs (Komiya *et al.*, 2008). In Arabidopsis, FD is redundant with FD PARALOG (FDP). Double *fd fdp* mutations flower later than *fd* single, and almost completely suppress precocious flowering of *35S:FT* (Jaeger *et al.*, 2013). In rice, by mutating additional bZIPs, including *OsFD3* and *OsFD4*, we have identified floral promoting factors. Mutations in *osfd4*, but not in *osfd3*, can delay flowering and activation of *OsMADS* targets. However, flowering was not delayed as much as in *osfd1* mutants, indicating a less prominent role compared to OsFD1. Also, *osfd1 osfd4* double mutants did not further delay flowering compared to
osfd1 single mutants. Based on expression data, we propose that OsFD1 activates OsFD4
transcription. Thus, in osfd1 mutants, the levels of both regulators are low. This arrangement does not
fully exclude redundancy but suggests a complex regulatory network.

Considering the interaction patterns with the florigens, we found that OsFD4 can interact directly
with RFT1, but only via Gf14s with Hd3a, suggesting different preferences for FAC architectures.
The SAM of rice is induced to flower by both florigens under SD, but only by RFT1 under LD
(Komiya et al., 2008, 2009). Since the flowering delay observed in osfd4 mutants is much stronger
under LD than under SD, we propose that different bZIPs perform distinct functions in the
photoperiod, and that the OsFD4-RFT1 module is key to promote flowering mostly under non-
inductive conditions.

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520 DAP-Seq identifies genome-wide OsFD1, OsFD4 and HBF1 binding sites

Application of DAP-seq using purified OsFD1, OsFD4 and HBF1 proteins resulted in the identification of hundreds of putative direct binding sites for these bZIPs. Consistent with a function in core promoters, binding sites were enriched in the proximity of the TSS. It should be noted that the *in vitro* nature of DAP-seq allows for the identification of direct binding events by a single TF, specifically capturing those mediated by individual homodimers. Given that OsFD1, OsFD4 and HBF1 formed heterodimers, these events may represent only a fraction of all *in vivo* binding events resulting from additional protein interactions. Integrating DAP-seq data with expression data allowed us to focus specifically on those genes that were expressed in relevant tissues and on those differentially expressed in the *osfd4-1* and *osfd1-2* mutants. Our data provide a first examination of the direct genome-wide binding landscape of bZIP flowering regulators in rice. This approach needs now to be refined for several reasons.

First, we used a single purified bZIP transcription factor in DAP-seq reactions. Being disengaged
from its florigen-containing complex, its DNA binding capacity might be biased. Arabidopsis FD can
bind functional sites without assembling into a FAC and with no need for FT or its closest homologue,
TWIN SISTER OF FT (TSF) (Collani *et al.*, 2019). Also purified OsFD1 alone can bind a fragment
of the *AP1* promoter containing a C-box element (GACGTC) in gel shift assays (Taoka *et al.*, 2011).
We previously showed that HBF1 can bind *in vitro* an ABRE/G-box element in the *Ehd1* promoter
region, a finding that we further corroborated in this study (Brambilla *et al.*, 2017). Thus, bZIPs are
likely to conserve their basal DNA binding ability even when not combined in ternary complexes,
but despite these examples, affinity for DNA is likely potentiated by binding with other interactors,
including members of the PEBP family (Collani *et al.*, 2019).
Second, since bZIPs bind the DNA as homo or heterodimers, and given their combinatorial interactions and overlapping expression in different plant tissues, including the SAM, it cannot be excluded that DNA binding specificity changes when assessing heterodimeric configurations.

Third, bZIPs might be incorporated into higher order complexes that include other transcriptional regulators with different DNA binding properties. In Arabidopsis, SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3), SPL4 and SPL5 interact with FD to enhance its transcriptional activation of target genes and its specificity for target promoters (Jung *et al.*, 2016). Since SPLs are produced at the SAM in response to plant aging, the SPL-FD complex has been proposed to integrate photoperiodic and aging signals to activate the *AP1* promoter (Jung *et al.*, 2016). The study further suggested that the proximal C-box in the *AP1* promoter is dispensable for FD-mediated induction, and that a distinct SPL-binding site might contribute to transcription of *AP1*. A similar case has been described for Class II TCP transcription factors, including TCP5, 13 and 17. These proteins can physically interact with FD, and facilitate its binding to the *AP1* promoter. Also in this case, binding of FD to the *AP1* promoter was not observed at the C-box, but was dependent upon a TCP binding motif located between the SPL-binding site and the C-box (Li *et al.*, 2019).

Finally, post transcriptional modifications have a major role *in vivo*. Many FD-like proteins harbour at their C terminus an SAP domain containing serine, threonine or both, which needs to be phosphorylated to interact with Gf14s (Taoka *et al.*, 2011; Park *et al.*, 2014; Li *et al.*, 2015; Collani *et al.*, 2019). When phosphorylated, Arabidopsis FD has a higher affinity for DNA. Expression of phosphomimic versions of FD or OsFD1 can accelerate flowering in Arabidopsis and rice, respectively. The kinases responsible for phosphorylation in Arabidopsis include Calcium-dependent Protein Kinase 6 (CPK6) and CPK33, which interact with FD and, if mutated, slightly delay flowering (Kawamoto *et al.*, 2015). Thus, optimal DNA binding and floral promoting activity of FAC complexes likely relies on this modification of the bZIP components. Future DNA-binding experiments either in vivo or using multiple complex components could help elucidate the individual contributions of each component.

## Direct targets of OsFD1 and OsFD4 and alternative binding syntaxes

Despite these caveats, DAP-seq led to the identification of several putative direct targets of the bZIPs studied here, out of which *OsMADS62* and *ARF19* were validated as transcriptionally regulated by both OsFD1 and OsFD4. *OsMADS62* controls pollen maturation and germination, partially redundantly with *OsMADS63* and *OsMADS68* (Liu *et al.*, 2013). OsFD1 and OsFD4 promoted *OsMADS62* expression, a function they likely perform after meristem commitment, during inflorescence and flower development. *OsARF19* encodes for an AUXIN RESPONSE FACTOR
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broadly expressed in the plant but with higher levels of transcription in the shoot, which is further
elevated by auxin treatments (Zhang et al., 2015).

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We did not find OsFD1 or OsFD4 bound to the *OsMADS14*, *OsMADS15* or *OsMADS34/PAP2* promoter. *OsMADS15* has been proposed as direct target of an OsFD1-containing FAC (Taoka *et al.*, 2011). Other proteins could possibly stabilize OsFD4 and OsFD1 *in vivo* and allow binding to these loci. Alternatively, the rice OsFD1-*OsMADS* connection might be indirect. Data in support of a direct connection have not been as thoroughly repeated and validated in rice as they have been in Arabidopsis, in which direct contacts between FD and the C-box element in the *AP1* promoter were disproved (Benlloch *et al.*, 2011; Jung *et al.*, 2016; Collani *et al.*, 2019; Li *et al.*, 2019). Further assessment of *in vivo* binding will be necessary.

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Genome scanning by DAP-seq identified identical core bZIP-binding motifs that were highly enriched in peaks of all three bZIP datasets. The same 5'-CACGT-3' core motif was identified as bound by OsFD1, OsFD4 and HBF1. The same 5'-CACGT-3' core motif was identified as enriched among FD binding regions *in vivo*, suggesting conservation between rice and Arabidopsis, and indeed many other bZIPs from diverse organisms (Dröge-Laser et al., 2018).

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We found that despite identical core DNA binding sites, spacing among tandem motifs was different for each of the three bZIPs tested in our assay. OsFD1 and OsFD4 which are activators of the rice floral transition prefer to bind the DNA with ER conformations, whereas HBF1, a repressor, binds more frequently ER and DR conformations. These results indicate distinct and specific binding syntaxes for each transcription factor. The method was recently applied to define binding syntaxes of MONOPTEROS (MP) and AtARF2, which have opposing transcriptional functions but share the same consensus motif (Stigliani *et al.*, 2019). The method can capture DNA binding features of proteins that interact as either homo or heterodimers, but the ER or DR arrangements might suggest the possibility of more elaborated structures, including tetramers.

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**Figure 1. The *osfd4* and *osfd1* mutants delay rice flowering.**

(a) Days to heading of *Dongjin* wild type (Dj) and *osfd4-1* under inductive short day conditions (SD) and non-inductive long day conditions (LD). (b) Days to heading of *Nipponbare* wild type (Nb), *osfd1-1*, *osfd3-1* and *osfd4-3* under SD conditions. (c) Representative pictures of *osfd1-1* (left) and Nb (right) plants. White arrows indicate emerging panicles. (d) Days to heading of Nb, *osfd1-2*, *Dj*, *osfd4-1* and double *osfd4-1 osfd1-2* mutants under SD conditions. Data are represented as mean ± StDev. Asterisks indicate the p-value calculated using ANOVA, ****=p<0.0001, **=p<0.01, ns=non significant. All flowering time experiments were repeated at least twice, and one representative experiment is shown in (a) and (b). The graph in (d) includes data from three independent experiments.

**Figure 2. Temporal expression pattern of *OsFD1*, *OsFD3* and *OsFD4* in the shoot apical meristem of rice and genetic interaction between *OsFD1* and *OsFD4*.

Quantification of *OsFD1*, *OsFD3* and *OsFD4* transcripts in the SAM (a) and in leaves (b) of Nb wild type. DAS, Days After Shift from long day (LD) to short day (SD) conditions. (c) Quantification of *OsFD4* transcripts in SAMs of *osfd1-1* mutants. (d) Quantification of *OsFD1* transcripts in SAMs of *osfd4-1* mutants. Each time point represents the mean ± StDev of three technical replicates. The experiments were repeated twice with similar results. Ubiquitin was used to normalize gene expression.

**Figure 3. Interactions between *OsFD1*, *OsFD3*, *OsFD4* and components of Florigen Activation Complexes (FACs) determined in yeast and tobacco.**

(a) Interactions between Activation Domain (AD)-OsFD1, AD-OsFD4 and Binding Domain (BD)-Gf14A-F. (b) Interactions between AD-Gf14A-F and BD-Hd3a and BD-RFT1. (c) Interactions between AD-OsFD4, AD-OsFD3 and Hd3a and RFT1 fused to the BD. Bimolecular fluorescence complementation (BiFC) between OsFD4 fused with N-terminus of YFP (N-YFP) and (d) RFT1 fused with the C-terminus of YFP (C-YFP) or (e) Hd3a fused with the C-YFP. (f) Interactions between OsFD4 and OsFD1 fused with the AD or the BD. (g) Interactions between OsFD3, OsFD4 and OsFD1 fused to the BD and OsFD3 fused to the AD. (h) Assessment of OsFDs homodimerization by BiFC. From left, OsFD4:N-YFP/OsFD4:C-YFP, OsFD1:N-YFP/OsFD1:C-YFP and OsFD3:N-YFP/OsFD3:C-YFP. (i) Assessment of OsFDs heterodimerization by BiFC. From left OsFD4:N-YFP/OsFD1:C-YFP, OsFD3:N-YFP/OsFD4:C-YFP and OsFD1:N-YFP/OsFD3:C-YFP.
634 Interactions were determined in yeast on selective drop out media -L-W-H-A. The yeast experiments 635 were repeated three times with identical results. AD and BD clones containing empty vectors were 636 used as negative controls. BiFC experiments were repeated three times in tobacco with identical 637 results. N-YFP and C-YFP clones containing empty vectors were used as negative controls. DAPI 638 stain was used to mark nuclei.

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640 **Figure 4. OsFD4 and OsFD1 promote OsMADS14 and 15 transcription in rice.**

641 Quantification of (a), (f) OsMADS14, (b), (g) OsMADS15, (c), (h) OsMADS18, (d), (i) OsMADS20, 642 and (e), (j) OsMADS34/PAP2 transcription in *Dongjin* vs. *osfd4-1* mutants (a)-(e) and in *Nipponbare* 643 vs. *osfd1-1* mutants (f)-(j). Expression was quantified in plants grown for two months under long day 644 (LD) and then shifted to short day (SD) conditions. Apical meristems were sampled at 0, 6, 12 and 645 18 Days After Shifting (DAS).

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647 **Figure 5. Identification of OsFD1, OsFD4 and HBF1 binding sites.**

648 (a) Summary of DNA Affinity Purification sequencing (DAP-seq) results of OsFD1, OsFD4 and 649 HBF1, including number of peaks, putative target genes and consensus motives. (b) Distribution of 650 peaks within gene features. (c) Distribution of peaks near the Transcriptional Start Site (TSS) of 651 putative target genes. (d) Venn diagrams showing the overlap between putative target genes of 652 OsFD1, OsFD4 and HBF1. (e) Venn diagrams showing the overlap between targets of OsFD1 and 653 OsFD4 and genes expressed at the apical meristem. (f) Venn diagrams showing the overlap between 654 targets of OsFD1 and HBF1 and genes expressed in leaves. Genome browser view of OsFD1 and 655 OsFD4 binding peaks in the 3'UTR of *OsMADS62* (g) and in the putative promoter of *OsARF19* (h), 656 and quantification of transcripts in *osfd4-1* and *osfd1-1* mutants. Black arrows indicate the direction 657 of transcription. Asterisks indicate p<0.01 (*), p<0.005 (**), p<0.001 (***) , p<0.0001 (****) based 658 on Student's t test, ns=non significant. Each time point represents the mean ± St Dev of three technical 659 replicates. The experiments were repeated twice with similar results. Ubiquitin was used to normalize 660 gene expression.

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662 **Figure 6. Analysis of OsFD1, OsFD4 and HBF1 binding syntax.**

663 (a) Scheme of Everted Repeats (ER), Direct Repeats (DR) and Inverted Repeats (IR) of “ACGTGGC” 664 motives. Blue and green arrows represent the first and the second binding motifs. N=general base; 665 n=number of base pairs between the two motifs. (b)-(d) Spacing analysis of absolute and normalized 666 enrichment of OsFD4 (b), OsFD1 (c) and HBF1 (d) binding sites. The numbers on top of the graphs 667 represent the distance between two consensus motives.
668

669 **Supplemental figures and tables**

670 **Fig. S1** Genotype and phenotype of *OsFD4* and *OsFD1* mutants

671 **Fig. S2** OsFD3 and Gf14s Y2H assays and OsFD4-Hd3a BiFC

672 **Fig. S3** Y2H mating controls

673 **Fig. S4** Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1

674 **Fig. S5** Expression analysis of some DAP-seq targets in *osfd4-1* and *osfd1-1*

675 **Fig. S6** Spacing analysis control: OsFD4, OsFD1 and HBF1 ROC curves

676 **Table S1** Sequences of oligonucleotides used in this study

677 **Table S2** Arabidopsis orthologs of rice genes

678 **Table S3** List of OsFD4, OsFD1 and HBF1 DAP-seq bound genes

679 **Table S4** List of genes shown in Figure S4

680

681 **Acknowledgements**

682 The authors wish to thank the NOLIMITS microscopy facility and Botanical Garden Città Studi for technical support. This work was supported by an ERC Starting Grant (#260963) and PRIN (#20153NM8RM) to FF.

685

686 **Author Contributions**

687 MC, MG, JL, F Parcy, AG, VB and FF designed research; MC, FG, MG, BK, JL and F Podico performed research; MC, FG, MG, BK, JL, ET, VB and FF analysed and interpreted data; MC and FF wrote the manuscript.

690

691 **REFERENCES**

692 **Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T. 2005.** FD, a bZIP Protein Mediating Signals from the Floral Pathway Integrator FT at the Shoot Apex. *Science* **309**: 1052–1056.

695 **Andrés F, Coupland G. 2012.** The genetic basis of flowering responses to seasonal cues. *Nature Reviews. Genetics* **13**: 627–39.

697 **Bartlett A, Malley RCO, Huang SC, Galli M, Nery JR, Gallavotti A, Ecker JR. 2017.** Mapping genome-wide transcription-factor binding sites using DAP-seq. *Nature Protocols* **12**: 1659–1672.

699 **Benlloch R, Kim MC, Sayou C, Thévenon E, Parcy F, Nilsson O. 2011.** Integrating long-day
700 flowering signals: a LEAFY binding site is essential for proper photoperiodic activation of
701 APETALA1. *The Plant Journal* **67**: 1094–1102.

702 Bolger AM, Lohse M, Usadel B. 2014. Genome analysis Trimmomatic: a flexible trimmer for
703 Illumina sequence data. *Bioinformatics* **30**: 2114–2120.

704 Brambilla V, Martignago D, Goretti D, Cerise M, Somssich M, de Rosa M, Galbiati F,
705 Shrestha R, Lazzaro F, Simon R, et al. 2017. Antagonistic Transcription Factor Complexes
706 Modulate the Floral Transition in Rice. *The Plant Cell* **29**: 2801–2816.

707 Cho L-H, Yoon J, Pasriga R, An G. 2016. Homodimerization of Ehd1 Is Required to Induce
708 Flowering in Rice. *Plant Physiology* **170**: 2159–2171.

709 Collani S, Neumann M, Yant L, Schmid M. 2019. FT Modulates Genome-Wide DNA-Binding of
710 the bZIP Transcription Factor FD. *Plant Physiology* **180**: 367–380.

711 Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A. 2004.
712 *Ehd1*, a B-type response regulator in rice, confers short-day promotion of flowering and controls
713 *FT*-like gene expression independently of *Hd1*. *Genes & Development* **18**: 926–36.

714 Dröge-Laser W, Snoek BL, Snel B, Weiste C. 2018. The Arabidopsis bZIP transcription factor
715 family — an update. *Current Opinion in Plant Biology* **45**: 36–49.

716 Du A, Tian W, Wei M, Yan W, He H, Zhou D, Huang X, Li S, Ouyang X. 2017. The DTH8-
717 Hd1 Module Mediates Day-Length-Dependent Regulation of Rice Flowering. *Molecular Plant* **10**:
718 948–961.

719 Furutani I, Sukegawa S, Kyozuka J. 2006. Genome-wide analysis of spatial and temporal gene
720 expression in rice panicle development. *The Plant Journal* **46**: 503–11.

721 Galbiati F, Chiozzotto R, Locatelli F, Spada A, Genga A, Fornara F. 2016. *Hd3a*, *RFT1* and
722 *Ehd1* integrate photoperiodic and drought stress signals to delay the floral transition in rice. *Plant*,
723 *Cell & Environment* **39**: 1982–1993.

724 Galli M, Khakhar A, Lu Z, Chen Z, Sen S, Joshi T, Nemhauser JL, Schmitz RJ, Gallavotti A.
725 2018. The DNA binding landscape of the maize AUXIN RESPONSE FACTOR family. *Nature
726 Communications* **9**: 4526.

727 Gómez-Ariza J, Brambilla V, Vicentini G, Landini M, Cerise M, Carrera E, Shrestha R,
728 Chiozzotto R, Galbiati F, Caporali E, et al. 2019. A transcription factor coordinating internode
729 elongation and photoperiodic signals in rice. *Nature Plants* **5**: 358–362.

730 Goretti D, Martignago D, Landini M, Brambilla V, Gómez-Ariza J, Gnesutta N, Galbiati F,

731 Collani S, Takagi H, Terauchi R, et al. 2017. Transcriptional and Post-transcriptional

732 Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes. *PLoS Genetics*

733 13:e1006530.

734 Hayama R, Yokoi S, Tamaki S, Yano M, Shimamoto K. 2003. Adaptation of photoperiodic

735 control pathways produces short-day flowering in rice. *Nature* **422**: 719–722.

736 Itoh H, Nonoue Y, Yano M, Izawa T. 2010. A pair of floral regulators sets critical day length for

737 Hd3a florigen expression in rice. *Nature Genetics* **42**: 635–8.

738 Izawa T, Foster R, Chua NH. 1993. Plant bZIP protein DNA binding specificity. *Journal of*

739 *Molecular Biology* **230**: 1131–1144.

740 Jaeger KE, Pullen N, Lamzin S, Morris RJ, Wigge PA. 2013. Interlocking feedback loops

741 govern the dynamic behavior of the floral transition in Arabidopsis. *The Plant Cell* **25**: 820–33.

742 Jang S, Li H-Y, Kuo M-L. 2017. Ectopic expression of Arabidopsis *FD* and *FD PARALOGUE* in

743 rice results in dwarfism with size reduction of spikelets. *Scientific Reports* **7**: 44477.

744 Jung J-H, Lee H-J, Ryu JY, Park C-M. 2016. SPL3/4/5 Integrate Developmental Aging

745 and Photoperiodic Signals into the FT-FD Module in Arabidopsis Flowering. *Molecular Plant* **9**:

746 1647–1659.

747 Kaneko-Suzuki M, Kurihara-Ishikawa R, Okushita-Terakawa C, Kojima C, Nagano-

748 Fujiwara M, Ohki I, Tsuji H, Shimamoto K, Taoka KI. 2018. TFL1-Like Proteins in Rice

749 Antagonize Rice FT-Like Protein in Inflorescence Development by Competition for Complex

750 Formation with 14-3-3 and FD. *Plant and Cell Physiology* **59**: 458–468.

751 Kawamoto N, Sasabe M, Endo M, Machida Y, Araki T. 2015. Calcium-dependent protein

752 kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen

753 complex formation. *Scientific Reports* **5**: 8341.

754 Kim S-K, Park H-Y, Jang YH, Lee KC, Chung YS, Lee JH, Kim J-K. 2016. OsNF-YC2 and

755 OsNF-YC4 proteins inhibit flowering under long-day conditions in rice. *Planta* **243**: 563–576.

756 Kobayashi K, Maekawa M, Miyao A, Hirochika H, Kyozuka J. 2010. PANICLE PHYTOMER2

757 (*PAP2*), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet
758 meristem identity in rice. *Plant and Cell Physiology* **51**: 47–57.

759 **Kobayashi K**, **Yasuno N**, **Sato Y**, **Yoda M**, **Yamazaki R**, **Kimizu M**, **Yoshida H**, **Nagamura Y**,
760 **Kyozuka J. 2012**. Inflorescence meristem identity in rice is specified by overlapping functions of
761 three *API/FUL*-like MADS box genes and *PAP2*, a SEPALLATA MADS box gene. *The Plant Cell*
762 **24**: 1848–59.

763 **Kojima S**, **Takahashi Y**, **Kobayashi Y**, **Monna L**, **Sasaki T**, **Araki T**, **Yano M. 2002**. *Hd3a*, a
764 rice ortholog of the Arabidopsis *FT* gene, promotes transition to flowering downstream of *Hd1*
765 under short-day conditions. *Plant & Cell Physiology* **43**: 1096–105.

766 **Komiya R**, **Ikegami A**, **Tamaki S**, **Yokoi S**, **Shimamoto K. 2008**. *Hd3a* and *RFT1* are essential
767 for flowering in rice. *Development* **135**: 767–774.

768 **Komiya R**, **Yokoi S**, **Shimamoto K. 2009**. A gene network for long-day flowering activates *RFT1*
769 encoding a mobile flowering signal in rice. *Development* **136**: 3443–3450.

770 **Langmead B**, **Salzberg SL. 2012**. Fast gapped-read alignment with Bowtie 2. *Nature Methods* **9**:
771 357–359.

772 **Li C**, **Lin H**, **Dubcovsky J. 2015**. Factorial combinations of protein interactions generate a
773 multiplicity of florigen activation complexes in wheat and barley. *The Plant Journal* **84**: 70–82.

774 **Li D**, **Zhang H**, **Mou M**, **Chen Y**, **Xiang S**, **Chen L**, **Yu D. 2019**. Arabidopsis Class II TCP
775 Transcription Factors Integrate with the FT–FD Module to Control Flowering. *Plant Physiology*
776 **181**: 97–111.

777 **Liu Y**, **Cui S**, **Wu F**, **Yan S**, **Lin X**, **Du X**, **Chong K**, **Schilling S**, **Theißen G**, **Meng Z. 2013**.
778 Functional Conservation of MIKC*-Type MADS Box Genes in *Arabidopsis* and Rice Pollen
779 Maturation. *The Plant Cell* **25**: 1288–1303.

780 **Liu B**, **Liu Y**, **Wang B**, **Luo Q**, **Shi J**, **Gan J**, **Shen WH**, **Yu Y**, **Dong A. 2019**. The transcription
781 factor OsSUF4 interacts with SDG725 in promoting H3K36me3 establishment. *Nature
782 Communications* **10**: 1–14.

783 **Miao J**, **Guo D**, **Zhang J**, **Huang Q**, **Qin G**, **Zhang X**, **Wan J**, **Gu H**, **Qu L-J. 2013**. Targeted
784 mutagenesis in rice using CRISPR-Cas system. *Cell Research* **23**: 1233–6.

785 **Nemoto Y**, **Nonoue Y**, **Yano M**, **Izawa T. 2016**. *Hd1*, a *CONSTANS* ortholog in rice, functions as
786 an *Ehd1* repressor through interaction with monocot-specific CCT-domain protein Ghd7. *The Plant*
787 *Journal* **86**: 221–233.

788 O'Malley RC, Huang S shan C, Song L, Lewsey MG, Bartlett A, Nery JR, Galli M, Gallavotti

789 A, Ecker JR. 2016. Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape.

790 *Cell* **166**: 1598.

791 Oliveros J. 2007. Venny. An interactive tool for comparing lists with Venn's diagrams. *Publicly*

792 available at http://bioinfogp.cnb.csic.es/tools/venny/index.html.

793 Park SJ, Jiang K, Tal L, Yichie Y, Gar O, Zamir D, Eshed Y, Lippman ZB. 2014.

794 Optimization of crop productivity in tomato using induced mutations in the florigen pathway.

795 *Nature Genetics* **46**: 1337–42.

796 Purwestri YA, Ogaki Y, Tamaki S, Tsuji H, Shimamoto K. 2009. The 14-3-3 protein GF14c acts

797 as a negative regulator of flowering in rice by interacting with the florigen Hd3a. *Plant and Cell*

798 *Physiology* **50**: 429–438.

799 Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Singla-Pareek SL. 2011. An improved protocol

800 for efficient transformation and regeneration of diverse indica rice cultivars. *Plant Methods* **7**: 49.

801 Stigliani A, Martin-Arevalillo R, Lucas J, Bessy A, Vinos-Poyo T, Mironova V, Vernoux T,

802 Dumas R, Parcy F. 2019. Capturing Auxin Response Factors Syntax Using DNA Binding Models.

803 *Molecular Plant* **12**: 822–832.

804 Supek F, Bošnjak M, Škunca N, Šmuc T. 2011. REVIGO Summarizes and Visualizes Long Lists

805 of Gene Ontology Terms (C Gibas, Ed.). *PLoS ONE* **6**: e21800.

806 Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K. 2007. Hd3a protein is a mobile

807 flowering signal in rice. *Science* **316**: 1033–1036.

808 Tamaki S, Tsuji H, Matsumoto A, Fujita A, Shimatani Z, Terada R, Sakamoto T, Kurata T,

809 Shimamoto K. 2015. FT-like proteins induce transposon silencing in the shoot apex during floral

810 induction in rice. *Proceedings of the National Academy of Sciences of the United States of America*

811 **112**: E901-10.

812 Taoka K, Ohki I, Tsuji H, Furuita K, Hayashi K, Yanase T, Yamaguchi M, Nakashima C,

813 Purwestri YA, Tamaki S, et al. 2011. 14-3-3 proteins act as intracellular receptors for rice Hd3a

814 florigen. *Nature* **476**: 332–5.

815 Teo C-J, Takahashi K, Shimizu K, Shimamoto K, Taoka K. 2017. Potato Tuber Induction is
816 Regulated by Interactions Between Components of a Tuberigen Complex. *Plant and Cell*
817 *Physiology* **58**: 365–374.

818 **Tian T, Liu Y, Yan H, You Q, Yi X, Du Z, Xu W, Su Z. 2017.** agriGO v2.0: a GO analysis
819 toolkit for the agricultural community, 2017 update. *Nucleic Acids Research **45**: W122–W129.

820 **Tsuji H, Nakamura H, Taoka K, Shimamoto K. 2013.** Functional diversification of FD
821 transcription factors in rice, components of florigen activation complexes. *Plant & Cell Physiology*
822 **54**: 385–97.

823 **Tylewicz S, Tsuji H, Miskolczi P, Petterle A, Azeez A, Jonsson K, Shimamoto K, Bhalerao RP.**
824 **2015**. Dual role of tree florigen activation complex component FD in photoperiodic growth control
825 and adaptive response pathways. *Proceedings of the National Academy of Sciences of the United
826 States of America **112**: 3140–5.

827 **Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU, Weigel D. 2005.**
828 Integration of spatial and temporal information during floral induction in Arabidopsis. *Science **309**:
829 1056–9.

830 **Wu F, Shi X, Lin X, Liu Y, Chong K, Theißen G, Meng Z. 2017.** The ABCs of flower
831 development: mutational analysis of *AP1/FUL*-like genes in rice provides evidence for a homeotic
832 (A)-function in grasses. *The Plant Journal **89**: 310–324.

833 **Xue W, Xing Y, Weng X, Zhao Y, Tang W, Wang L, Zhou H, Yu S, Xu C, Li X, et al. 2008.**
834 Natural variation in *Ghd7* is an important regulator of heading date and yield potential in rice.
835 *Nature Genetics **40**: 761–767.

836 **Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K,**
837 **Umehara Y, Nagamura Y, et al. 2000.** *Hd1*, a major photoperiod sensitivity quantitative trait locus
838 in rice, is closely related to the Arabidopsis flowering time gene *CONSTANS*. *The Plant Cell **12**:
839 2473–2484.

840 **ZHANG S, WANG S, XU Y, YU C, SHEN C, QIAN Q, GEISLER M, JIANG DA, QI Y. 2015.**
841 The auxin response factor, OsARF19, controls rice leaf angles through positively regulating *OsGH
842 3-5* and *OsBRI 1*. *Plant, Cell & Environment **38**: 638–654.

843 **Zhao J, Chen H, Ren D, Tang H, Qiu R, Feng J, Long Y, Niu B, Chen D, Zhong T, et al. 2015.**
844 Genetic interactions between diverged alleles of *Early heading date 1* (*Ehd1*) and *Heading date 3a*
845 (*Hd3a*)/*RICE FLOWERING LOCUS T1* (*RFT1*) control differential heading and contribute to
846 regional adaptation in rice (Oryza sativa). *New Phytologist* **208**: 936–948.

847 **Zhu S, Wang J, Cai M, Zhang H, Wu F, Xu Y, Li C, Cheng Z, Zhang X, Guo X, et al.** 2017.

848 The OsHAPL1-DTH8-Hd1 complex functions as the transcription regulator to repress heading date

849 in rice. *Journal of Experimental Botany* **68**: 553–568.
<figure><img src="image_3.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<caption>(a)</caption>

SAM

<figure><img src="image_7.png" /><figcaption>(b)</figcaption></figure>

LEAVES

<figure><img src="image_8.png" /><figcaption>(c)</figcaption></figure>

Nb wt osfd1-1

<figure><img src="image_9.png" /><figcaption>(d)</figcaption></figure>

Dj wt osfd4-1

<figure><img src="image_10.png" /></figure>
<caption>(a)</caption>

BD::Gf14A

BD::Gf14B

BD::Gf14C

BD::Gf14D

BD::Gf14E

BD::Gf14F

BD empty
vector

AD::OsFD1

AD::OsFD4

AD empty
vector

<figure><img src="image_11.png" /></figure>

-W, -L, -H, -A

<caption>(b)</caption>

AD::Gf14A

AD::Gf14B

AD::Gf14C

AD::Gf14D

AD::Gf14E

AD::Gf14F

AD empty
vector

<figure><img src="image_12.png" /></figure>

-W, -L, -H, -A

BD::Hd3a

BD::RFT1

BD empty
vector

<caption>(c)</caption>

AD::OsFD4

AD::OsFD3

AD empty
vector

BD::Hd3a

BD::RFT1

BD empty
vector

<figure><img src="image_13.png" /></figure>

-W, -L, -H, -A

<caption>(d)</caption>

OsFD4:N-YFP
RFT1:C-YFP

<figure><img src="image_14.png" /><figcaption>(e)</figcaption></figure>

OsFD4:N-YFP
Hd3a:C-YFP

<figure><img src="image_15.png" /><figcaption>(f)</figcaption></figure>

BD::OsFD4

BD::OsFD1

BD empty
vector

AD::OsFD4

AD::OsFD1

AD empty
vector

<figure><img src="image_16.png" /></figure>

-W, -L, -H, -A

<caption>(g)</caption>

BD::OsFD3

BD::OsFD4

BD::OsFD1

BD empty
vector

AD::OsFD3

AD empty
vector

<figure><img src="image_17.png" /></figure>

-W, -L, -H, -A

<caption>(h)</caption>

OsFD4:N-YFP
OsFD4:C-YFP

<figure><img src="image_18.png" /></figure>

OsFD1:N-YFP
OsFD1:C-YFP

<figure><img src="image_19.png" /></figure>

OsFD3: N-YFP
OsFD3:C-YFP

<figure><img src="image_20.png" /><figcaption>(i)</figcaption></figure>

OsFD4:N-YFP
OsFD1:C-YFP

<figure><img src="image_21.png" /></figure>

OsFD3:N-YFP
OsFD4:C-YFP

<figure><img src="image_22.png" /></figure>

OsFD1:N-YFP
OsFD3:C-YFP

<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>(f)</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>(g)</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>(h)</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>(i)</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>(j)</figcaption></figure>

<figure><img src="image_33.png" /></figure>
<table><caption>(a)</caption><thead><tr><th>bZIP</th><th>Nr of peaks</th><th>Nr of putative target genes</th><th>Motif</th></tr></thead><tbody><tr><td>HBF1</td><td>28323</td><td>15937</td><td><img src="motif1.png" alt="Motif 1"></td></tr><tr><td>OsFD1</td><td>2059</td><td>1717</td><td><img src="motif2.png" alt="Motif 2"></td></tr><tr><td>OsFD4</td><td>1107</td><td>925</td><td><img src="motif3.png" alt="Motif 3"></td></tr></tbody></table>

<figure><img src="image_34.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>(f)</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>(g)</figcaption></figure>

OsMADS62

<figure><img src="image_39.png" /><figcaption>(h)</figcaption></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>
<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>
**New Phytologist Supporting Information**

**Article title:** OsFD4 promotes the rice floral transition via Florigen Activation Complex formation in the shoot apical meristem

Authors: Martina Cerise, Francesca Giaume, Mary Galli, Bahman Khahani, Jérémy Lucas, Federico Podico, Elahe Tavakol, François Parcy, Andrea Gallavotti, Vittoria Brambilla and Fabio Fornara

Article acceptance date: 15 July 2020

The following Supporting Information is available for this article:

**Fig. S1** Genotype and phenotype of OsFD4 and OsFD1 mutants

**Fig. S2** OsFD3 and Gf14s Y2H assays and OsFD4-Hd3a BiFC

**Fig. S3** Y2H mating controls

**Fig. S4** Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1

**Fig. S5** Expression analysis of some DAP-seq targets in osfd4-1 and osfd1-1

**Fig. S6** Spacing analysis control: OsFD4, OsFD1 and HBF1 ROC curves

**Table S1** Sequences of oligonucleotides used in this study

**Table S2** Arabidopsis orthologs of rice genes (see separate file)

**Table S3** List of OsFD4, OsFD1 and HBF1 DAP-seq bound genes (see separate file)

**Table S4** List of genes shown in Figure S4 (see separate file)
<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

WT

+ 16

- 4

+ 1

367

<figure><img src="image_56.png" /></figure>

WT

+ 1

- 1

<figure><img src="image_57.png" /></figure>

WT

- 2

- 1

<figure><img src="image_58.png" /><figcaption>**Fig. S1** (a) Map of the *osfd4-1* mutant locus. Green and blue lines represent exons and UTR regions, respectively. A black line indicates an intron. A triangle in the 3'UTR region indicates the position of the T-DNA insert. (b) Expression of *OsFD4* in the SAM of Dj wild type and *osfd4-1* mutant. Data are average ± StDev of three technical replicates. (c) Sequences of mutant alleles of *osfd4*, *osfd1* and *osfd3* obtained using CRISPR. Inserted nucleotides are indicated in green. (d) Protein alignment of the C-terminus region of OsFD1, OsFD4 and OsFD3 starting from the basic leucine zipper domain. (e) Flowering time under LD conditions of *osfd4-3* and *osfd4-4*.</figcaption></figure>
Data are represented as mean ± StDev. Asterisks indicate the p-value calculated using ANOVA,
***=p<0.001, **=p<0.01.

<figure><img src="image_59.png" /></figure>

**Fig. S2** (a) Yeast two Hybrid (Y2H) among Gf14A-F fused to the Binding Domain (BD) and OsFD3 fused to the Activation Domain (AD). (b) Double entry table summarizing homo- and heterodimerizations among Gf14A-F. Interactions were assessed on drop out media lacking L, W, H and A. N.T., not tested. (c) Bimolecular fluorescence complementation (BiFC) between OsFD4 fused with C-terminus of YFP (C-YFP) and Gf14B fused with the N-terminus of YFP (N-YFP). (d) BiFC between empty C-YFP vector and OsFD4 fused with N-YFP (left); Hd3a fused with C-YFP and N-YFP empty vector (right). Negative controls were done for every independent BiFC assay performed; here representative ones are shown.
<figure><img src="image_60.png" /><figcaption>**Fig. S3** (a) Mating controls of the Y2H among Gf14A-F fused to the AD and Hd3a or RFT1 fused to the BD. (b) Mating controls of the Y2H among Gf14A-F fused to the BD and OsFD1, OsFD4 and OsFD3 fused to the AD. (c) Mating controls of the Y2H among OsFD4 and OsFD3 fused to the AD and Hd3a and RFT1 fused to the BD. (d) Mating controls of the Y2H among OsFD4 and OsFD1 fused with the AD or the BD. (e) Mating controls of the Y2H among OsFD3, OsFD4 and OsFD1 fused to the BD and OsFD3 fused to the AD. All controls were grown on yeast medium lacking W and L.</figcaption></figure>
<figure><img src="image_61.png" /><figcaption>**Fig. S4** Venn diagram showing the overlap between targets of AtFD, OsFD1, OsFD4 and HBF1.</figcaption></figure>
<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

**Fig. S5** Quantification of transcripts of LOC_Os01g14440 (WRKY) (a), LOC_Os01g64360 (MYB) (b), LOC_Os04g51000 (RICE FLORICAULA/LEAFY) (c), LOC_Os04g31730 (B3) (d) and
LOC_Os07g41580 (NF-YB) (e) in osfd4-1 and osfd1-1 mutants. Data are represented as mean ± StDev.

<figure><img src="image_72.png" /><figcaption>**Fig. S6** Receiver Operating Characteristics (ROC) analysis of OsFD4 (a), OsFD1 (b) and HBF1 (c) estimating the prediction power of Position Weight Matrices (PWMs), i.e. the capability of OsbZIPs binding sites to differentiate between bound and not bound regions. AUC, Area Under the Curve.</figcaption></figure>
<table><caption>Table S1 Sequences of oligonucleotides used in this study.</caption><thead><tr><td>Number</td><td>Sequence</td><td>Gene</td></tr></thead><tbody><tr><td>Os_892</td><td>GACAACGTGAAGGCGAAGA</td><td>UBQ Fw for qRT-PCR</td></tr><tr><td>Os_893</td><td>CACCAGGTGGAGTGTGGAC</td><td>UBQ Rv for qRT-PCR</td></tr><tr><td>Os_245</td><td>CCGGGCTATGGAGTGAAAT</td><td>OsFD4 Fw for qRT-PCR</td></tr><tr><td>Os_246</td><td>TGCTCTCATATTCTCCATGCTG</td><td>OsFD4 Rv for qRT-PCR</td></tr><tr><td>Os_160</td><td>TTCAGGTGGACGACCTTAGC</td><td>OsFD1 Fw for qRT-PCR</td></tr><tr><td>Os_161</td><td>GCTAGCAGCTGCCAACACT</td><td>OsFD1 Rv for qRT-PCR</td></tr><tr><td>Os_158</td><td>CGGTTGCGAGACGAGGAA</td><td>OsMADS14 Fw for qRT-PCR</td></tr><tr><td>Os_159</td><td>GAAAGACGGTGCTGGACGAA</td><td>OsMADS14 Rv for qRT-PCR</td></tr><tr><td>Os_239</td><td>CGTCGTCGGCCAAACAG</td><td>OsMADS15 Fw for qRT-PCR</td></tr><tr><td>Os_240</td><td>TGACTTCAATTCATTCAAGGTTGCT</td><td>OsMADS15 Rv for qRT-PCR</td></tr><tr><td>Os_353</td><td>TGTTCTGCAAAAGCTCATGG</td><td>OsMADS18 Fw for qRT-PCR</td></tr><tr><td>Os_354</td><td>TTGGTGATGATGTGCTTGGT</td><td>OsMADS18 Rv for qRT-PCR</td></tr><tr><td>Os_1353</td><td>GCTCCAGTCATCATCGAACG</td><td>OsMADS20 Fw for qRT-PCR</td></tr><tr><td>Os_1354</td><td>GGAGATACTACTACCGCTCACT</td><td>OsMADS20 Rv for qRT-PCR</td></tr><tr><td>Os_186</td><td>TTGATGAACTCTGCGACCTAAA</td><td>OsMADS34 Fw for qRT-PCR</td></tr><tr><td>Os_187</td><td>TGCTGCAGTTTCCGTTCC</td><td>OsMADS34 Rv for qRT-PCR</td></tr><tr><td>Os_1312</td><td>AGAGGGGCAACTTGAGGAAC</td><td>OsARF19 Fw for qRT-PCR</td></tr><tr><td>Os_1313</td><td>GCAACCGACAAATTCCTCC</td><td>OsARF19 Rv for qRT-PCR</td></tr><tr><td>Os_1341</td><td>CAGTACCTGTCGATGGAGCA</td><td>OsMADS62 Fw for qRT-PCR</td></tr><tr><td>Os_1342</td><td>GTTGCTGTCGTACGCCATG</td><td>OsMADS62 Rv for qRT-PCR</td></tr><tr><td>Os_1349</td><td>GCAACACCAACAACACAAGC</td><td>LOC_Os01g14440 Fw for qRT-PCR</td></tr><tr><td>Os_1350</td><td>AGTGTATACCATGTGCCCACT</td><td>LOC_Os01g14440 Rv for qRT-PCR</td></tr><tr><td>Os_1351</td><td>GCATCCATGACATCACCACC</td><td>LOC_Os01g64360 Fw for qRT-PCR</td></tr><tr><td>Os_1352</td><td>ATCAGAGAGCCCCAAGTGAC</td><td>LOC_Os01g64360 Rv for qRT-PCR</td></tr><tr><td>Os_1369</td><td>GTTCAGGGTCAGGTCCTC</td><td>LOC_Os04g31730 Fw for qRT-PCR</td></tr><tr><td>Os_1370</td><td>ACCTGAAACTGAAACCTCCCA</td><td>LOC_Os04g31730 Rv for qRT-PCR</td></tr><tr><td>Os_1371</td><td>ATGGTGATGGGAGGGAAAGG</td><td>LOC_Os07g41580 Fw for qRT-PCR</td></tr><tr><td>Os_1372</td><td>ACCCGACATGTCCTCTGATT</td><td>LOC_Os07g41580 Rv for qRT-PCR</td></tr><tr><td>Os_1373</td><td>GTACGTGCCCACCAGACT</td><td>LOC_Os04g51000 Fw for qRT-PCR</td></tr><tr><td>Os_1374</td><td>GCCCAACACCACAGGAAAC</td><td>LOC_Os04g51000 Rv for qRT-PCR</td></tr><tr><td>Os_1151</td><td>GGCACCGCCGGCGTCGTTTCAGGA</td><td>Oligo CRISPR OsFD4 Fw</td></tr><tr><td>Os_1152</td><td>AAACTCCTGAAACGACGCCGGCGG</td><td>Oligo CRISPR OsFD4 Rv</td></tr><tr><td>Os_936</td><td>GGCATCTTGATCATCCGCCTCTTG</td><td>Oligo CRISPR OsFD1 Fw</td></tr><tr><td>Os_937</td><td>AAACCAAGAGGCGGATGATCAAGA</td><td>Oligo CRISPR OsFD1 Rv</td></tr><tr><td>Os_1153</td><td>GGCAGCCCACGCCGAGGGATAGAT</td><td>Oligo CRISPR OsFD3 Fw</td></tr><tr><td>Os_1154</td><td>AAACATCTATCCCTCGGCGTGGGC</td><td>Oligo CRISPR OsFD3 Rv</td></tr></tbody></table>