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Transcriptional and Post-transcriptional Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes

Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.

Introduction Rice (Oryza sativa L.) was originally domesticated~8000 years ago in tropical Asia. Archeological remains and genome re-sequencing indicated southern China as the region of first cultivation, despite the debate regarding the domestication dynamics is still open [1][2][3]. Although of tropical origin, rice is currently cultivated across a broad latitudinal range [4,5]. Expansion to temperate areas required selection of varieties better adapted to local environmental conditions. Tolerance to low temperatures and day length-insensitive flowering have been crucial adaptive traits under selection.
Flowering (or heading date) is the result of an intricate series of pathways that mediate between environmental inputs and the production of molecules inducing flowering, known as florigens. Photoperiod is the major environmental cue that rice plants utilize to measure seasonal time [6]. Rice, as well as other important cereals including sorghum and maize, is a short day (SD) plant in which flowering is induced as the duration of the light phase during the day does not exceed a critical threshold. Under such inductive conditions, expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1), florigenic genes highly similar to Arabidopsis FLOWERING LOCUS T (FT), is induced in the vascular tissue of leaves [7][8][9][10][11]. Expression of the florigens is triggered by EARLY HEADING DATE 1 (Ehd1) encoding a Btype response regulator protein central to the photoperiodic flowering network [12]. The Ehd1 gene is unique to rice and its transcription is strongly controlled at diurnal and seasonal levels [12,13]. Mapping of QTLs identified several major regulators of Ehd1 expression that, upon cloning, were shown to encode transcription factors belonging to distinct protein groups [14][15][16][17][18][19][20]. In particular, GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) and PSEUDO RESPONSE REGULATOR 37 (PRR37) encode CCT (CONSTANS, CONSTANS-like, TOC1) domain proteins, whereas Ghd8 encodes the NF-YB11 subunit of the NF-Y transcription factor complex [14][15][16]: all of them encode strong floral repressors. Artificial selection of rice varieties adapted to grow in Europe or Asia has taken advantage of loss-of-function alleles at such loci, because de-repression of Ehd1 expression results in up-regulation of the florigens and subsequent flowering also under non-inductive day lengths [14][15][16]21,22]. Sensitivity to day length can be compromised to the extent that pyramiding of specific mutations completely abolishes it [23,24].
A major repressor of Ehd1 transcription is encoded by HEADING DATE 1 (Hd1), a zincfinger CCT-domain transcription factor, homologous to CONSTANS of Arabidopsis [10,23,25,26]. As opposed to CO that promotes flowering under inductive long days (LD), Hd1 performs a dual function, because under LD conditions it delays flowering, whereas under SD conditions it promotes it by inducing expression of the florigens [10,27]. Similarly to floral repressor genes already mentioned, extensive allelic variation has been described at the Hd1 locus that includes a plethora of loss-of-function alleles associated to varieties adapted to a broad latitudinal range [21,23,[28][29][30][31]. However, the dual molecular function of Hd1 and the modes of repression of florigenic loci under LD are poorly understood. Although extensive genetic variation for flowering time traits has been described, more allelic variants must exist within local germplasm collections, because described genetic diversity appears insufficient to fully account for reduced sensitivity to day length of all varieties. Additionally, most efforts have focused on the identification of polymorphisms creating clear loss-of-function mutations, such as frame shifts or premature stops codons. Such first level of investigation provides an important but limited description of standing variation and additional levels have to be explored.
Comparisons between rice and Arabidopsis can help to derive and test hypotheses concerning protein function, albeit these two species diverged~150M years ago and the extent of conservation of the Hd1-Hd3a/CO-FT modules is debated [32,33]. In Arabidopsis, CO can interact with several AtNF-YB and AtNF-YC subunits [34,35]. Induction of FT expression and flowering mediated by CO requires some of these subunits, because nf-yb2 nf-yb3 or nf-yc3 nf-yc4 nf-yc9 mutants fail to induce FT expression and flower late under LD [35,36]. Additionally, the early flowering phenotype of plants overexpressing CO from a strong promoter is limited by the double nf-yb2 nf-yb3 or triple nf-yc3 nf-yc4 nf-yc9 mutations [35,37]. The CO protein can directly bind the proximal promoter of FT in vivo [38] or CO Response Elements (CORE) in vitro [37]. Whether the recruitment of CO to the FT promoter is enhanced by the interaction with NF-YB and NF-YC subunits upon formation of a CO-containing NF-Y heterotrimeric complex is currently unknown. It is similarly unclear if rice relies on a trimeric NF-Y system to regulate expression of the florigens and flowering.
In this study, we used varieties flowering at higher latitudes to identify novel polymorphisms at loci relevant for photoperiodic adaptation. Two novel and common Hd1 alleles were found, both sufficient to create a non-functional variant. Taking advantage of such genetic tools, we hypothesized and demonstrated the formation of a NF-Y heterotrimeric complex containing Hd1, capable of binding to a conserved response element in the Hd3a promoter. Genetic variation at Hd1 can impinge on trimer formation and the floral transition. Our results suggest how multiple layers of variation can be stratified at the same locus and independently exploited during artificial selection. Additionally, they show how genetic diversity can provide unique molecular variants to understand specific developmental processes at the molecular level.

Results
The Hd1 EH allele promotes rice flowering under long days Artificial selection of loss-of-function mutations in floral repressor genes, including Hd1, Ghd7, Ghd8 and PRR37 has been an effective strategy to expand rice cultivation to higher latitudes in both Asia and Europe [4,23,28,39,40]. However, the genetic determinants that allowed expansion have not been fully determined for all varieties and additional major regulators or novel haplotypes not previously described are likely to be present in several accessions. A genetic approach was used to identify candidate genes conferring reduced sensitivity to day length. A segregating population was obtained by crossing Nipponbare (NB) with Erythroceros Hokkaido (EH), a temperate japonica variety from Poland [29]. The genome of NB harbors functional Hd1, Ghd7, Ghd8 and PRR37 that confer sensitivity to day length (Photoperiod Sensitivity Index, PSI = 0.69), whereas EH is insensitive to the photoperiod (PSI = 0.16) and flowers very early regardless of external light conditions (Fig 1). Flowering of the resulting F2 individuals was scored under LD conditions (16L/8D) and followed a normal distribution ( Fig  1A). The EH parental line was examined with molecular markers designed on known mutant alleles of floral repressors, revealing the presence of homozygous ghd7-0a and prr37-2a loss-offunction genes [14,16,23,40]. Consistently with this finding, the earliest flowering F2 plants cosegregated with these mutant alleles (S1 Fig). The Hd1 locus of EH (Hd1 EH ) was sequenced, including the coding region (CDS), introns and untranslated 5' and 3' regions and was found to be identical to haplotype Hd1-VII [23]. The Hd1 EH allele apparently encodes for a functional protein, based on the absence of indels creating frame shifts or premature stop codons in the CDS. However, it co-segregated with very early heading plants, effectively behaving as a nonfunctional LD repressor or as constitutive activator of flowering ( Fig 1A). To distinguish the effects of ghd7, prr37 and Hd1 EH on flowering, F3 plants bearing single or multiple mutations were selected and heading dates were scored under natural long days (NLD) in Milan (45.47˚N) (S2 Fig). Under NLD, the Hd1 EH allele strongly promoted flowering and pyramiding of prr37 further accelerated it, indicating additive effects. Combinations of prr37 and ghd7 produced the shortest cycle length (S2 Fig). To assess the differences between Hd1 NB and Hd1 EH we selected F3 lines carrying wild type Ghd7 and PRR37 alleles and scored heading dates under increasing photoperiods ( Fig 1B). Phenotypic differences between photoperiodic treatments were mild in lines harboring the Hd1 EH allele compared to lines harboring the Hd1 NB allele, confirming that genotypes containing Hd1 EH have reduced sensitivity to day length.

Transcription of Hd1 is suppressed in Erythroceros Hokkaido
To understand the functional effects of Hd1 EH on flowering, the mRNA levels of downstream targets of Hd1 were quantified under LD conditions in selected F3 genotypes harboring the Hd1 EH or Hd1 NB alleles. The Hd1 EH allele caused precocious transcription of the florigens, and particularly that of RFT1, compared to plants carrying the Hd1 NB allele (Fig 2C and 2D). Expression of Ehd1 was also elevated in plants bearing the Hd1 EH allele ( Fig 2B). As Hd1 represses Ehd1 under LD conditions [23], increased transcription of Ehd1 could be explained by reduced functionality or expression of Hd1. Interestingly, expression of Hd1 from plants  harboring the Hd1 EH allele was almost undetectable during the entire time course under LD (Fig 2A), a feature not previously described for any Hd1 allele. Transcript abundance of Hd1 shows a diurnal rhythm and to assess if low levels of These data indicate that Hd1 EH is never expressed and it can be considered a loss-of-function allele. Silencing of Hd1 is therefore an effective strategy to promote heading and indicates the existence of a tunable layer of variation creating phenotypic diversity.

A mobile element is responsible for suppression of Hd1 transcription in several varieties
Regulatory elements in the Hd1 promoter could be responsible for variation of its transcription. To test this hypothesis, fourteen varieties were chosen from the European Rice Core Collection (ERCC) that harbored functional alleles of Ghd7, Ghd8 and PRR37 and three distinct alleles at the Hd1 locus, including Hd1 NB , Hd1 EH and Hd1 from Volano (Hd1 Vol ), a widely cultivated, high-yielding variety from Italy [41]. About 1.2 Kb of DNA upstream of the ATG was sequenced in this panel and all varieties carrying the Hd1 EH alleles were found to contain a sequence of~4.4Kb at position -166bp, annotated as mobile element (GenBank accession AB300057.1) (Fig 3A). Whether this DNA sequence has features of a transposable or retrotransposable element is unclear. However, some sequences in the mobile element are expressed (Genbank accession AK101779.1). An additional copy of this same element is present in the rice genome, on chromosome 6. Hd1 was expressed in all varieties except those bearing the mobile element ( Fig 3A). Using diagnostic primers, 242 varieties belonging to the ERCC were screened and 92 (38%) were identified that contained the mobile element (S1 Table). The same screen performed on a world panel, including 77 varieties belonging to all Oryza genetic groups, identified only two bearing the mobile element, suggesting that the non-expressed allele is more widely distributed among European varieties (S2 Table).
Among all varieties of the ERCC bearing functional copies of Hd1, Ghd7, Ghd8 and PRR37, as defined by Gómez-Ariza et al. (102 accessions in total), two groups were distinguished based on the presence or absence of the mobile element (S1 Table). Expression of Hd1 was quantified in 4-week-old seedlings grown under LD. Transcript levels were undetectable in varieties bearing the promoter insertion, whereas expression of Ghd7 and PRR37 was similar between the two groups ( Fig 3B-3D). To clear the effects of the genetic background, we cloned the Hd1 promoters of NB and EH, and used them to drive expression of the ß-glucuronidase gene (GUS) upon transformation into NB and EH calli. The pHd1 NB >>GUS vector was active in both the NB and EH calli, whereas the pHd1 EH >>GUS could not produce expression spots in EH and NB calli, indicating that failure to express the reporter was caused by cis-acting elements in pHd1 EH (Fig 3E). Taken together, these data indicate that insertion of a mobile element in the Hd1 promoter prevents its expression. This is sufficient to reduce sensitivity to day length in several varieties and adapt them to European regions. These results also show how a transposable element has been instrumental to human selection to spread cultivation of a major cereal at higher latitudes, and add to the prominent roles that transposons have played during domestication and later diversification of crops [42][43][44][45].

QTL-Seq identifies the Hd1 Vol allele and associates it with early heading under long days
Of the 102 varieties belonging to the ERCC and categorized as having functional copies of LD floral repressors [23], 64 silence expression of Hd1 through a mobile element inserted in its regulatory regions. From the remaining pool that expresses Hd1, Volano (Vol) was selected and crossed with NB to produce a recombinant population suitable for QTL mapping. An F2 segregating progeny comprising 138 individuals was grown under controlled LD (16L/8D) and heading dates were scored ( Fig 4A). A normal distribution for days to heading was observed with several plants flowering very late and showing transgressive segregation. The DNA of twenty individuals from the earliest and latest flowering plants was bulked separately and a QTL-Seq approach was applied to identify the loci responsible for heading date variation [46]. A strong peak in the ΔSNP score was detected with high statistical significance on chromosome 6, representing the major locus controlling flowering in this cross ( Fig 4B). The QTL corresponded to the position of the Hd1 locus ( Fig 4B). The normal distribution for days to heading suggested the existence of additional genes. Two QTLs were identified on chromosome 1 and 10 in which the NB allele promoted and delayed flowering, respectively. However, their statistical significance was lower compared to the QTL on chromosome 6, possibly due to low sequencing coverage and/or dominance effects of the QTLs (S4 Fig) [46].
The coding sequence of Hd1 Vol includes two in-frame insertions, several non-synonymous and conservative substitutions, and the deletion of a Lysine in the CCT domain, when compared to Hd1 NB (S5 Fig). Therefore, it is not interrupted by deletions or frame shifts and based on genetic evidences, it could be hypothesized that impaired function is caused by abolished mRNA expression, altered protein activity or mislocalization. Quantification of mRNA showed that Hd1 transcripts abundance was reduced, but not abolished in Volano when compared to NB [23], and among the flowering repressors, also Ghd7 and PRR37 transcripts showed reduced diurnal cycling amplitude (S6 Fig). We transiently expressed the Hd1 NB -GFP and Hd1 Vol -GFP proteins in tobacco leaves under an inducible promoter and observed that both protein variants accumulated after induction and were targeted to the nucleus (S7 Fig). These data indicate that Hd1 Vol is expressed, cycles normally and is targeted correctly. We therefore tested whether the polymorphisms in Hd1 Vol prevent interactions with relevant partners binding to target genes. A heterotrimeric NF-Y transcription complex can accommodate Hd1 NB but not Hd1 Vol The AtNF-YB2/3 and AtNF-YC3/4/9 proteins are necessary for FT expression and flowering of Arabidopsis [34][35][36]. NF-YB and NF-YC form a histone fold domain (HFD) scaffold that accommodates NF-YA, the sequence-specific subunit of the CCAAT-binding trimer NF-Y [47][48][49]. Binding of AtNF-Y to the CCAAT box in the distal promoter of FT controls its expression and HFDs were shown to interact with CO [37,50,51]. In rice the corresponding components are encoded by Hd1, Ghd8 (OsNF-YB11) and NF-YC subunits. Whether a NF-Y trimeric complex formed in rice and artificial selection of polymorphisms in single components affected features of the complex, including protein-protein interactions or DNA binding properties, has never been addressed. Assembly of a trimeric complex by distinct NF-Y components was therefore tested using yeast-two and three-hybrid assays. First, transcripts of the seven NF-YC genes encoded in the rice genome were quantified, to select for those expressed in leaves where the complex is likely formed. Transcripts of NF-YC3 and NF-YC5 were not expressed under LD conditions (S8 Fig); the remaining genes were all expressed at similar levels, except NF-YC2, whose diurnal oscillations were wider both under LD and SD (S8 Fig). The NF-YC1, NF-YC2 and NF-YC7 proteins were then expressed in yeast together with Ghd8. Heterodimeric interactions were observed for all combinations, as well as Ghd8 homodimerization (S3 Table). No direct interactions were detected between Hd1 NB or Hd1 Vol and NF-YC1, NF-YC7 or Ghd8. Therefore, we could not reproduce recent data indicating interaction between Hd1 and Ghd8, possibly because different cultivars were used [52]. As both BD:NF-YC2 and BD:Hd1 fusion proteins could autoactivate the yeast reporters, their interaction could not be determined.
The NF-YB and NF-YC subunits form the histone fold domain scaffold that accommodates the third subunit of the trimeric complex. Using yeast-three-hybrid assays, a strong interaction was observed between Hd1 NB /NF-YC1/Ghd8 and Hd1 NB /NF-YC7/Ghd8. However, Hd1 Vol could not interact with the NF-YC1/Ghd8 or NF-YC7/Ghd8 heterodimers (Table 1), indicating that some polymorphisms in Hd1 Vol prevent the formation of the heterotrimer.

OsPRR37 can form an alternative OsNF-Y heterotrimer
OsPRR37 is a major LD repressor whose CCT-domain shows homology to the CCT of Hd1 and structural homology to NF-YA [16,40,53]. To address the combinatorial properties of the rice NF-Y complex, OsPRR37 was used in a yeast-three-hybrid assay together with NF-YC subunits and Ghd8. Growth of yeast on selective media indicated that the OsPRR37 protein could interact with the NF-YC1/Ghd8 and NF-YC7/Ghd8 heterodimers (Table 1). Taken together, these data indicate that (i) a trimeric complex can be assembled between Hd1, Ghd8 and distinct NF-YC subunits, among which at least NF-YC1 and NF-YC7 interact strongly within the trimer, (ii) genetic variation creates protein variants unable to interact with the HFD and (iii) OsPRR37 (and possibly other PRR proteins) can replace Hd1 in the heterotrimer.
Hd1 NB but not Hd1 Vol can bind a response element present in the promoter of Hd3a The formation of a NF-Y trimer shown above and the resemblance of the CCT domain to the NF-YA domain required for CCAAT-binding [53], suggest that the CCT of Hd1 could impart sequence-specificity to the trimer, as well as being sufficient for heterotrimerization [34,50]. We assessed the DNA-binding properties of the complex by electrophoretic mobility shift assays (EMSA). We produced the HFDs of OsNF-YC7 and Ghd8, and the CCT domains of Hd1 NB and Hd1 Vol in E. coli. Note that CCT-Hd1 NB and CCT-Hd1 Vol differ only for a lysine, missing in Vol and lying in the first part of the CCT domain. This region, based on structural homology with the NF-YA A1 helix [53,54], is involved in protein-protein interactions with HFD proteins and highly conserved among CO-like proteins in monocots and dicots (S5 Fig). The DNA probe was selected within the Hd3a proximal promoter region, based on the presence and conservation of a CO Response Element 2 (CORE2) located at -169bp [37,51]. Fig 4C shows that a shifted band was detected when CCT-Hd1 NB /Ghd8/OsNF-YC7 were incubated together, reconstituting a trimeric complex. The band shift was not observed in the presence of CCT-Hd1 Vol or when Ghd8/OsNF-YC7 was missing (Fig 4C). To check for specificity, we challenged the complex with an unlabeled oligonucleotide identical to the DNA probe, or containing mutations in the CORE2 site (Fig 4D). The wild type oligonucleotide, but not the mutated one, competed binding efficiently. Overall, these data corroborate the yeast analysis indicating that Hd1 NB forms a trimer with the OsNF-Y HFDs, and confirm that Hd1 Vol , defective in HFDs association, is unable to bind a CORE2 element in DNA-binding assays.
Finally, we checked the distribution of the Hd1 ΔK337 polymorphism in the ERCC and found a total of 38 varieties sharing this mutation (Fig 4E). Notably, 33 of these belonged to the subset of 102 accessions mentioned above (S1 Table). Thus, the large majority of expressed variants of Hd1 have been likely selected because they compromise the repressor function of the complex.

Discussion
An OsNF-Y protein complex links transcriptional regulators within the photoperiodic pathway The genetic architecture of the rice photoperiodic pathway heavily relies on floral repressor genes encoding transcription factors. These have been the first components to be isolated by genetic mapping in the flowering regulatory network and include Hd1, Ghd7, Ghd8 and PRR37 [14,16,25,[55][56][57]. The position of such genes in the network initially suggested the existence of separate regulatory branches having partly unrelated effects. The Ghd7 and Ghd8 mutants have been isolated as independent regulators of Ehd1 [15,55]. Mutations in PRR37 have been initially believed to repress flowering by limiting Hd3a expression but not that of Ehd1 or RFT1 [16]. A later study indicated that PRR37 acts upstream of both florigens by controlling Ehd1 expression [40]. Until recently, the pathways centered on Hd1 and Ehd1 have been considered independent and acting in parallel, but recent data established a connection between Hd1 activity and Ehd1 expression, demonstrating Hd1 to be an upstream repressor of Ehd1 under LD [23]. The data presented in this study indicate that Hd1, Ghd8 and PRR37 proteins do not act independently but rather assemble into a higher-order NF-Y protein complex that constitutes the molecular core of the photoperiodic pathway (Fig 5A). The recent demonstration of a molecular interaction between Hd1 and Ghd7 proteins at the Ehd1 promoter, despite not directly implicating a heterotrimeric complex, further corroborates this interpretation [58]. Finally, binding of the heterotrimer to an element present in the Hd3a promoter suggests the existence of multiple targets for the OsNF-Y complex within the flowering network.

Combinatorial properties of the OsNF-Y complex in the regulation of flowering time in rice
The NF-Y complex is a sequence-specific heterotrimeric transcription factor formed by histone-like subunits and common to eukaryotes [59]. However, whereas in animals and fungi each component of the complex is encoded by a single gene, plant genomes have largely amplified the number of subunits and in species such as rice or Arabidopsis hundreds of combinations of the NF-YA, B and C subunits are possible, that fine tune the spatio-temporal regulation of gene expression while enormously expanding the range of regulated processes [48,[60][61][62].
In rice, the major NF-YB subunit involved in flowering time regulation is Ghd8 (OsN-F-YB11), however, other components, including OsNF-YB10 and OsNF-YB8 share high sequence similarity with Ghd8 and might have a role as regulators of flowering in rice [53]. This view is corroborated by the fact that late flowering of Arabidopsis nf-yb2 nf-yb3 double mutants is rescued by expression of OsNF-YB10 or OsNF-YB8, suggesting an effect on flowering time control, at least in a heterologous system [63]. Additionally, overexpression of OsN-F-YB7 and OsNF-YB9 delays flowering in rice under LD conditions [64]. Despite their sequence being only weakly related to that of Ghd8, they might compete with Ghd8 in the complex that includes it, or form an alternative one. Therefore, although Ghd8 is a prominent regulator, the existence of other NF-YB subunits regulating flowering in the Hd1/PRR37 containing complexes cannot be ruled out and deserves further attention.
Based on yeast interactions and DNA-protein binding assays we showed that OsNF-YC1 and OsNF-YC7 can interact with Ghd8 and CCT-domain proteins including, at least, Hd1 and PRR37. Trimer formation with OsNF-YC2 and OsNF-YC4 could not be formally demonstrated because of auto-activation of yeast reporters. However, dimeric interactions between OsNF-YC2 and OsNF-YB8, OsNF-YB10 and OsNF-YB11 have been reported [65]. Most importantly, genetic evidences support a role for OsNF-YC2 in the control of flowering in rice [65]. Transcriptional silencing of OsNF-YC2 by RNA-interference (RNAi) resulted in accelerated flowering under LD conditions, whereas its overexpression under the maize ubiquitin promoter strongly delayed flowering [65]. A milder effect on LD repression of flowering has been reported for OsNF-YC4, whereas OsNF-YC6 seems to have no role in flowering time control [65]. Taken together, these data indicate that in rice cells multiple HFD scaffolds can form and possibly bind to NF-YA or CCT domain proteins to control heading.
Demonstration of a heterotrimeric interaction between HFD dimers and PRR37 indicates for the first time that the HFD scaffold can bind proteins different from NF-YA or CO and CO-related proteins, all of which share a structurally similar, albeit not identical, CCT domain [34]. These findings further expand the combinatorial properties of the complex and might suggest a competitive mode of assembly, whereby Hd1 or other related proteins, including PRR-like or Ghd7-like factors, dynamically replace each other while interacting with the HFDs. Such model has been previously proposed for the CO2 and VRN2 proteins that were shown to compete with each other for binding to NF-YA, B or C components of wheat [47], but could be much more diversified among plant species as more CCT interactors become implicated in trimer formation. Additional combinations could be provided by direct interactions between CCT domain proteins. Recently, a direct interaction between Hd1 and Ghd7 was reported, and the Ghd7 protein was shown to bind the Ehd1 promoter [58]. Whether Hd1 or other NF-Y subunits are required for Ghd7 binding to DNA is still unclear. Also, how the dynamical assembly of proteins around Hd1 is regulated is unknown. The NFYB/C dimers and Ghd7 could compete with each other for interacting with Hd1, similarly to CO2 and VRN2 wheat proteins, either at specific times of the day or season. Alternatively, Hd1 could be the scaffold on which both HFD proteins and Ghd7 interact, forming a large and unique LD repressor complex.
Since DNA specificity is determined by NF-YA or CCT domain proteins, a further layer of variation is provided by the sequences bound by such components, possibly being the CCAAT box [50], CORE elements ( [37,51] and R.M., N.G. personal communication) or morning elements [66]. Finally, a crucial issue to address is when or in which cells an Hd1-containing complex is predominant over a PRR-containing complex to regulate expression of Ehd1, Hd3a or other genes, and how the dynamics of assembly and activity of alternative complexes are regulated at diurnal or seasonal levels. Not secondary to this question is the fact that since PRR proteins are central components of plants circadian clocks [67], the rhythm of expression of several genes other than those involved in flowering time control, might be dependent upon specific higher-order NF-Y complexes. Tissue-specific and temporal patterns of expression of NF-Y genes could help distinguishing between complexes possibly involved in regulation of photoperiodic responses (that take place in the vascular tissue of leaves or in cells of the shoot apex) from those involved in circadian clock functions (that take place in most cell types) [35,61].

The photoperiodic pathways of rice and Arabidopsis share similarities and differences
Based on recent findings and on the results presented in this study, it is worth reconsidering the comparison between the photoperiodic regulatory networks of rice and Arabidopsis.
Day length responses in rice are not controlled by distinct pathways but by a unique one, whose regulatory elements converge on Ehd1 [12]. Homologs of Ehd1 have not been identified in Arabidopsis or other dicot species, but they are present in the genomes of monocots, thus encoding a function not shared by all plants, and that likely evolved after the split between monocots and dicots about 150M years ago [68,69]. The gene works as an upstream transcriptional activator of Hd3a and RFT1 and promotes flowering under SD also in the absence of a functional Hd1 [12,70]. However, its repression under LD is mediated by genes whose homologs are present in Arabidopsis, and function in the regulation of flowering time also in dicot species.
Similarly to Arabidopsis, the OsGI, Hd1, Hd3a/RFT1 genetic cascade is present in rice as well.
The origin of the CO function and the conservation of the CO-FT module across Angiosperms has been challenged [32]. Simon et al., proposed that a CO function has evolved in the Brassicaceae family only after the most recent genome duplication that occurred within the family and that is not shared by its sister family [33]. Evolution of such function created a flexible switch to trigger flowering under LD. According to this interpretation, the Hd1 function might have evolved by convergent evolution. Consistent with a distinct origin (and distinct environmental pressures of tropical vs temperate areas), it is to be considered that a major function of Hd1 is to repress flowering under LD, and this function seems prominent compared to its function as SD flowering activator. These functions are not shared by Arabidopsis CO and the repressive activity in particular is directed to Ehd1 [23,58]. Therefore, the Ehd1 function seems to have been added to, or co-evolved with an existing network containing homologs that are shared with Arabidopsis, and that Ehd1 became central to the photoperiodic pathway of rice, as well as a hub gathering signals also from other environmental cues [71].
The DNA binding assays performed with heterotrimeric complexes indicates that the Hd1-containing NF-Y complex has the capacity to bind a CORE2 element from the Hd3a promoter. Similar assays have demonstrated that CO can bind the FT promoter and that CORE sequences are necessary for binding [37]. Thus, protein-DNA interactions between Hd1-Hd3a and CO-FT suggest the existence of similar regulatory modules in rice and Arabidopsis. However, since the CO function evolved only recently in the Brassicaceae and the Hd1 function evolved by convergent evolution, the existence of such modules and their similar arrangement is striking [33]. This might be indicative of their robustness at the core of the photoperiodic pathway. The Hd1 protein could bind also to the Ehd1 proximal promoter, as shown by chromatin immunoprecipitation assays, although no CORE sites have been identified in such region [58]. These data point to a three-node coherent feed forward loop of regulation under LD, directly linking Hd1 to Ehd1 and Hd3a (Fig 5B and 5C). This mechanism might have evolved because the presence of an Ehd1 floral inductive function unlinked from an Hd1 repressive function could have resulted in the induction of Hd3a/RFT1 expression also under LD. However, with both Hd3a and Ehd1 under direct control of Hd1, this problem would be overcome and long photoperiods would prevent flowering by limiting all floral activators. The role of RFT1 in such feed forward loop remains to be addressed. However, searches for CORE elements resulted in the identification of additional sites in both Hd3a and RFT1 loci. Follow up studies in vivo will test if these can be effective binding sites for NF-Y repressor complexes.

Plant material and growth conditions
The Japanese rice variety Nipponbare (NB, Hd1 Ghd7 PRR37 Ghd8 hd6) was crossed with Erythroceros Hokkaido (EH, Hd1 EH ghd7 prr37 Ghd8 Hd6) to produce a recombinant F2 population comprising 215 individuals. Depending on the genotype, F2 individuals were selected and heading dates were determined using F3 plants under different photoperiodic conditions. Volano (Vol, Hd1 Vol Ghd7 PRR37 Ghd8), a high-yielding variety from Italy, was crossed with NB and heading dates of 138 F2 individuals were scored.
The core collection comprising 242 varieties cultivated in Europe has been already described in [23]. Details of the accessions and genotypes of Hd1, Ghd7, PRR37 and Ghd8 are available in S1 Table. Plants were grown under controlled conditions in Conviron PGR15 chambers or greenhouses under LD (16h light) or SD (10h light) photoperiodic regimes. Day and night temperatures were 28˚C and 24˚C, respectively. Humidity was set at 70% during the day and~90% during the night.
Field experiments were performed at the Botanical Garden Città Studi, Milan (45.47˚N). Seeds were sown in a cold greenhouse on Apr 11, 2014 and transplanted in an irrigated field on May 17, 2014. Heading dates were scored from~30 plants/genotype. The photoperiod sensitivity index (PSI) was calculated as in [23].

Preparation of genomic DNA, polymerase chain reaction and sequencing
Genomic DNA was prepared from leaves using a modified CTAB and chloroform:isoamyl alcohol method [72]. Genotyping of the NBxEH F2 population was performed using markers for the prr37-2a and ghd7-0 alleles [23], whereas distribution of the Hd1 NB and Hd1 EH alleles was determined using primers listed in S4 Table. Genomic DNA was amplified using LA Taq from TaKaRa in Buffer I, according to manufacturer's indications. For each PCR reaction, DNA was initially incubated five minutes at 95˚C, followed by 40 cycles of amplification (95˚C 30 seconds 58˚C 30 seconds and 72˚C 1 minute). The same PCR profile was applied to all PCR reactions, extending or shortening the extension time depending on the expected fragment size.
By using the same PCR conditions, the European rice varieties were screened for the presence of the 4.4Kb mobile element in the Hd1 promoter, using forward 5'-promoter-anchored and reverse 3'-promoter-anchored primers in combination with primers designed within the mobile element. Additionally, a pair designed around the insertion site that could amplify only in the absence of the mobile element was used (S4 Table). Sequencing reactions were prepared and analyzed according to [23].

RNA extraction and quantification of mRNA abundance
Total RNA was extracted using the TRI Reagent (Sigma Aldrich) from the distal part of young leaves collected from at least three independent plants. Genomic DNA was digested using TURBO DNAse (Life Technologies) and the RNA was precipitated with sodium acetate and ethanol and resuspended in water. After quantification of total RNA, 1μg was retrotranscribed with SuperScriptII Reverse Transcriptase (Invitrogen) and oligo-dT according to manufacturers' instructions. The cDNA product was diluted 10 fold with sterile water. Transcripts were quantified in a Realplex 2 (Eppendorf). Reactions were carried out using 3μl of cDNA as template, 5μl of 2X Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and 0.2μl of each primer (final concentration 10μM) and ddH 2 O to a final volume of 10μL. A list of primers used for mRNA quantification is available in S4 Table. In particular, primers used to detect Hd1 expression are located in the 3'UTR region, that was sequenced and found to be identical between Hd1 EH and Hd1 NB , excluding the possibility that the primers used could not detect one of the two allelic variants.

Transient expression in tobacco leaves
The coding sequence of Hd1 NB was fused at the C-terminus with mCherry and that of Hd1 Vol was fused with GFP in pABind vectors [73]. Expression of the fusion proteins was under a β-estradiol inducible promoter. Tobacco leaves were infiltrated with Agrobacterium cultures containing the plasmids. A 20μM β-estradiol solution was sprayed on leaves 3-12 hours before observation of epidermal cells using a confocal microscope.

Yeast-two-and three-hybrid experiments
The coding sequences of genes used in yeast two-and three-hybrid assays were amplified from cDNA prepared from mature leaves using primers listed in S4 Table. The full length clone of Ghd8 was synthesized by GENEWIZ Inc. (South Plainfield, NJ) whereas OsPRR37 and NF-YC4 clones were obtained from the Rice Genome Resource Center (http://www.rgrc. dna.affrc.go.jp/index.html.en). All genes were cloned in pDONR207 (Life Technologies). Each entry clone was recombined with pGADT7 and pGBKT7 (Clontech), to obtain AD-and BDfusion proteins.
The AH109 and Y187 strains were used in yeast transformation as described in the Clontech manual for the Matchmaker Gold yeast-two-hybrid system. Transformed cultures were selected on YSD media lacking leucine (Leu), tryptophan (Trp) or adenine (Ade) for pGADT7, pGBKT7 and pTFT1, respectively. Protein-protein interactions were assessed by streaking colonies on YSD media lacking Leu, Trp and Histidine (His) for Y2H experiments and on media lacking Leu, Trp, Ade and His for Y3H experiments. The strength of the interactions was evaluated by streaking colonies on increasing amounts of 3-aminotriazole (3AT). Yeast growth was verified after 6 days at 30˚C. Each experiment has been repeated at least 3 times using independent clones.

QTL mapping
The QTL-Seq approach has been previously described [46]. Briefly, DNA was prepared using the C-TAB method to extract genomic DNA individually from the twenty earliest and twenty latest flowering plants, within a total population of 138 F2s. DNA was quantified and two DNA pools of early and late flowering plants were produced using 1μg of genomic DNA per each plant. The whole genome was re-sequenced using Illumina HiSeq 2500 with chemistry v4 at Eurofins (Germany), producing 125bp paired ends reads. Whole-genome resequencing yielded 18896 and 24961Mbp, with an approximate coverage of 39 and 52 fold for the early and late flowering pools, respectively. Filtered short reads were aligned to the NB reference genome and SNP indexes were calculated for the early and late heading bulks. SNP values of less than 0.3 in both samples were removed and a ΔSNP-index was determined and plotted on the chromosome maps. Finally, using a sliding window analysis candidate QTLs were visualized.

Protein purification
The cDNAs encoding the HFD of Ghd8 and OsNF-YC7 were synthesized by Eurofins Genomics and subcloned in pmcnCS EATCH using NdeI/BamHI restriction sites (S5 Fig). Only Ghd8 was tagged with 6x-His [74]. The CCT domains of Hd1 NB and Hd1 Vol were synthesized by Eurofins Genomics. The resulting proteins were tagged with 6x-His at the C-terminus and subcloned in pmcnEATCH. Soluble HFD heterodimers were produced in E. coli by co-expression in BL21 (DE3) strains by IPTG induction, and purified using the HisSelect resin (SIGMA). The CCT domains of Hd1 NB and Hd1 Vol were purified separately. Proteins were eluted in Buffer A (10mM Tris pH8.0, 400mM NaCl, 2mM MgCl 2 ), containing 250mM imidazole. Purified proteins were dialyzed against Buffer B (10mM Tris-Cl pH8.0, 400mM NaCl, 2mM DTT, 10% glycerol).

Promoter analysis using GUS histochemical assays
The promoter regions of NB (580bp) and EH (4996bp including the mobile element) were amplified with primers suitable for Gateway cloning and recombined into pDONR207 vectors (Life Technologies). Positive clones were confirmed by sequencing and recombined into pBGWFS7, to drive expression of a GFP-GUS reporter gene, using LR clonase (Life Technologies).
Embryogenic calli of NB and EH were derived from scutella of mature seeds. For callus induction, seeds were dehusked, sterilized and placed on basal NB-medium plates (pH5.8), supplemented with 3mg/L 2,4D, 0.25mg/L cytochine and 30g/L glucose, for three weeks in the dark and 28˚C. Proliferating embryogenic calli were subcultured on fresh medium for another three weeks before biolistic transformation.
Four hours before the bombardment and 16 hours after, calli were transferred on NB osmotic medium containing 34.6g/L of both mannitol and sorbitol, and they were kept in the dark at 28˚C. After the osmotic treatment, calli were placed on basal MS medium supplemented with 3mg/L 2,4 D for two days before GUS staining. Non-bombarded calli and calli bombarded with gold microcarries only were used as negative controls.
After bombardment, calli were incubated in the dark at 28˚C for at least two days. The viable calli were then incubated in 90% cold acetone at -20˚C for 20', washed twice with NaPO 4 buffer and finally transferred into the GUS histochemical reagent containing 1mM K 4 Fe (CN) 6 , 1mM K 3 Fe(CN) 6 , 0,1M NaPO 4 , 10mM EDTA, 0,1x Triton, 2mM X-Gluc. Samples were vacuum infiltrated for 20' and then incubated for 24h at 37˚C. After staining, samples were cleared with 70% ethanol.  Table. HFD formation between Ghd8 and OsNF-YC subunits assessed by yeast-2-hybrid. Proteins were expressed in yeast as fusions to the GAL4 binding domain (in pGBKT7) or to the GAL4 activation domain (in pGADT7). Interactions were assessed on diploid yeast after mating between AD and BD clones, as represented. The strength of the interaction was quantified adding 3-amino-triazole (3AT) at increasing concentrations. A-sign indicates no interaction in plates that do not contain 3AT, +++ indicates strong interaction in plates containing 20mM 3AT. n.t., not tested. Interactions between two clones was not always tested upon swapping AD and BD domains. (XLSX) S4 Table. List of primers used in this study. (XLSX)