Introduction

NUCLEAR FACTOR Y transcription factors [NF-Y, also known as heme-activator protein (HAP) and CCAAT box binding factors (CBFs)] regulate diverse genes in all eukaryotes (Edwards et al. 1998; Maity and de Crombrugghe 1998; Laloum et al. 2013). Individual NF-Y subunits identified in plant species are also known to play an essential role in the regulation of developmental processes (Meinke et al. 1994; Li et al. 2008; Yamamoto et al. 2009; Petroni et al. 2012; Laloum et al. 2013; Mu et al. 2013). NF-YB and NF-YC subunits are found as CONSTANS (CO)-interacting proteins in tomato and Arabidopsis thaliana (Ben-Naim et al. 2006; Wenkel et al. 2006). The AtNF-YB2 and AtNF-YB3 genes are known to promote flowering through the activation of FLOWERING LOCUS T (FT) under long-day (LD) conditions in Arabidopsis (Cai et al. 2007; Chen et al. 2007; Kumimoto et al. 2008). The AtNF-YC subunit genes also have functional redundancy in the promotion of flowering through the activation of FT under LD condition in Arabidopsis (Kumimoto et al. 2010). In addition, recent systematic studies in Arabidopsis have revealed that physical interaction between AtNF-YB and AtNF-YC is required for the formation of the entire heterotrimeric complex, prior to binding to the consensus CCAAT motif, as shown for mammalian cells (Calvenzani et al. 2012; Hackenberg et al. 2012). The AtNF-YC3, AtNF-YC4, and AtNF-YC9 proteins are also known to interact with the AtNF-YB2, AtNF-YB3, and CO subunits (Kumimoto et al. 2010), suggesting that the AtNF-YC subunits make heterotrimeric complexes with AtNF-YB2 or AtNF-YB3 and CO to provide unique platforms for the fine-tuning of flowering under various environmental conditions (Wenkel et al. 2006; Kumimoto et al. 2010). Contrary to the canonical NF-Y complexes, CO/AtNF-YB/AtNF-YC trimeric complexes are speculated not to bind the CCAAT motif in the FT promoter, because the CO protein does not have the essential histidine residues required for NF-Y complex binding (Xing et al. 1993; Siefers et al. 2009; Kumimoto et al. 2010). Furthermore, it was proposed that the canonical AtNF-Y complex binds a CCAAT motif within the FT distal enhancer and recruits CO to the proximal FT promoter elements (Petroni et al. 2012).

Our understanding of the roles of rice (Oryza sativa) NF-Y transcription factors in the control of flowering is still very limited. A rice protein, homologous to AtNF-YB2 (OsNF-YB11/OsHAP3H/Ghd8/DTH8), has recently been shown to regulate both flowering and grain yields (Wei et al. 2010; Yan et al. 2011). In addition, Ghd7/OsI (encoding a CCT domain-containing protein, Griffiths et al. 2003) regulates flowering under LD conditions (Xue et al. 2008). Although protein–protein interactions between some subsets of OsNF-YA/OsHAP2 or OsNF-YB/OsHAP3 and OsNF-YC/OsHAP5 have been reported in yeast (Thirumurugan et al. 2008), the interactions between OsNF-Y proteins and rice CO homologues such as Heading date 1(Hd1) have not been established. In wheat (Triticum aestivum), CO2 (a rice Hd1 homolog) and flowering repressor VRN2 (ZCCT1 and 2) compete for interactions with TaNF-Y complexes to integrate the vernalization and photoperiodic signals (Li et al. 2011). In addition, the Brachypodium distachyon NF-YB3 (BdNF-YB3) and BdNF-YB6 could rescue the late flowering phenotype of Arabidopsis nf-yb2 nf-yb3 double mutant and also interact with AtNF-YC3, AtNF-YC4, and AtNF-YC9 in yeast (Cao et al. 2011). These data suggest that homologous NF-Y transcription factors form heterotrimeric complexes, which regulate flowering time in monocot plants.

In this study, we investigated the function of rice proteins homologous to AtNF-YC3, AtNF-YC4 and AtNF-YC9. We report here that OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) interact with OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11) and the OsNF-YC proteins affect flowering time under LD conditions in rice.

Materials and methods

Plant materials and growth conditions

Wild-type rice (Oryza sativa L., cv. Hwaan; a gift from National Institute of Agricultural Biotechnology, Suwon, South Korea) and transgenic rice plants (in the O. sativa L., cv. Hwaan background) were grown in a growth chamber at 28 °C under either short-day (SD) conditions [10/14 h (light/dark)] or long-day (LD) conditions [14.5/9.5 h (light/dark)] with light supplied at an intensity of 100 µmol m−2 s−1, as described in Kim et al. (2008). For growth under SD conditions, rice plants were first grown in LD conditions for 20 days and then transferred to SD conditions until flowering. The flowering time was measured as the days to heading (DTH), which is the number of days from planting in pots to the emergence of the first panicle from the flag leaf sheath, as described previously (Kim et al. 2008).

Generation of transgenic plants

To generate green fluorescence protein (GFP)-tagged OsNF-YC overexpressing plants, the coding sequences of OsNF-YC2, OsNF-YC4 and OsNF-YC6 were amplified using specific primers and with each cDNA clone as the template and cloned in pCAMBIA1300 or pCAMBIA2300 binary vectors. Subsequently, the open-reading frames (ORFs) of three OsNF-YC including GFP sequences were amplified and inserted into the pGA1611 binary vector with the maize Ubi1 promoter. To generate RNAi transgenic plants, the OsNF-YC ORF sequences, for which inverted repeats were made, were amplified using specific primers, cloned into the pDONR201 vector (Invitrogen, Carlsbad, CA, USA), and subsequently recombined into the pANDA vector (a gift from Dr. Ko Shimamoto, NAIST, Japan). Oligonucleotide sequences used for cloning are provided in Supplementary Table S1. Agrobacterium-mediated rice transformation was performed, as described by Hiei and Komari (2008). Regenerated plants were transferred to soil and grown in a greenhouse for 1 month. The transformants were then tested for herbicide resistance by treating leaves with a 0.5 % Basta solution. Insertion of the introduced transgenes into the rice genome and their expression were confirmed by polymerase chain reaction (PCR) analysis.

RNA expression analysis

For real-time quantitative polymerase chain reaction (RT-qPCR) analysis, total RNA was extracted from adult leaves using QIAzol Lysis reagent (Qiagen, Hilden, Germany) and first-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA in accordance with the manufacturer’s instructions (Invitrogen). The RT-qPCR analysis was carried out in 384-well plates with a LightCycler 480 (Roche Applied Science, Madison, WI, USA) using SYBR green. RT-qPCR experiments were carried out using LightCycler 480 SYBR Green Master mixture (Roche Applied Science). All RT-qPCR experiments were performed in three technical triplicates. Two or three biological replicates were performed for transgenic plants. Oligonucleotide sequences used for expression analysis are provided in Supplementary Table S1. Changes in gene expression were calculated via the ∆∆CT method (Ramakers et al. 2003).

Protein expression analysis

Rice leaves from transgenic lines were ground to a powder in liquid nitrogen and the powder was suspended in a buffered solution of 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 % glycerol, 0.5 % Triton X-100, 2 mM phenylmethanesulfonyl fluoride (PMSF) and complete protease inhibitor cocktail (Roche Applied Science). The protein concentration was determined using the Bradford solution (Bio-Rad, Hercules, CA, USA). Subsequently, proteins were separated by SDS-PAGE onto a 12.5 % gel, transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and detected by Western blot, as described previously (Kim et al. 2008).

In vitro protein–protein interaction analysis

For in vitro glutathione S-transferase (GST) pull-down assays, the ORFs of OsNF-YB8, OsNF-YB10, OsNF-YB11, OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6 and OsNF-YC7 were cloned into the pGEX-5X-1 (GE Healthcare, LC, UK) to express GST fusion proteins in Escherichia coli. To generate in vitro translated products, the respective ORFs were cloned into the pET21a-d(+) vector (GE Healthcare), and in vitro translation products were synthesized using the T7 TNT-coupled Transcription/Translation System (Promega, Fitchburg, WI, USA). GFP-tagged OsNF-YB ORFs were cloned into the pET21a-d (+) expression vector (GE Healthcare). Oligonucleotide sequences used for cloning are provided in Supplementary Table S1. The detailed procedure has been previously reported (Jang et al. 2009).

Subcellular localization and bimolecular fluorescence complementation (BiFC) analyses

The ORFs of Ghd7, Hd1, and OsCO3, OsNF-YB8, OsNF-YB10, OsNF-YB11, OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6 and OsNF-YC7 were cloned into pCAMBIA1300 or pCAMBIA2300 for the localization assay and were also cloned into pSPYNE-35S or pSPYCE-35S for the BiFC assay (Walter et al. 2004). Oligonucleotide sequences used for cloning are provided in Supplementary Table S1. Agrobacterium strain C58C1 was used for infiltration of tobacco (Nicotiana benthamiana). Subsequently, epidermal cells of infiltrated tobacco leaves were examined for fluorescence using a confocal microscope (LSM 510 META, Carl Zeiss, Jena, Germany).

Results

OsNF-YC2, OsNF-YC4 and OsNF-YC6 proteins interact with OsNF-YB8, OsNF-YB10 and OsNF-YB11 proteins

AtNF-YC3, AtNF-YC4 and AtNF-YC9 are known to regulate flowering time via protein–protein interaction with AtNF-YB2, AtNF-YB3 and CO proteins in Arabidopsis (Kumimoto et al. 2010). OsNF-YB11 (rice AtNF-YB2 and AtNF-YB3 homologs) functions in the control of heading date in rice (Wei et al. 2010; Yan et al. 2011). Five OsNF-YC proteins (OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6 and OsNF-YC7) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11) of rice were identified as AtNF-YC3/4/9 and AtNF-YB2/3 homologous proteins, respectively (Thirumurugan et al. 2008). We tested if these rice proteins are functional homologs to Arabidopsis counterparts. GST pull-down assays were performed to examine the interactions between the OsNF-YC proteins and OsNF-YB proteins. OsNF-YB8 and OsNF-YB11 proteins interacted with five OsNF-YC proteins. Compared with the intensity of the 10 % input, two OsNF-YB proteins showed more significant interactions with two of the OsNF-YC proteins (OsNF-YC6 and OsNF-YC7) than with the other three OsNF-YC proteins (OsNF-YC1, OsNF-YC2 and OsNF-YC4) (Fig. 1a). Protein–protein interactions between OsNF-YB10 protein and five OsNF-YC proteins were not determined because it was not possible to produce the OsNF-YB10 protein for this assay.

Fig. 1
figure 1

Protein–protein interaction between three OsNF-YB proteins and five OsNF-YC proteins in vitro. a GST pull-down assay between two OsNF-YB proteins and five OsNF-YC proteins. GST or GST-tagged OsNF-YC proteins were incubated with 35S-labeled OsNF-YB8 or 35S-labeled OsNF-YB11 proteins. The bands on the gel images indicate the eluted OsNF-YB8 or OsNF-YB11 proteins as visualized by autoradiography. The last lane of each panel shows 10 % of the 35S-labeled proteins added to each pull-down reaction mixture. Coomassie blue-stained bands (CBB) indicated by asterisks show the amount and quality of the GST fusion proteins used in this assay. b GST pull-down assay between GST-tagged OsNF-YC proteins and OsNF-YB proteins tagged with both GFP and His. The eluates were separated by 12.5 % SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with anti-His antibody (α-His). About 4 % of the total sample in each reaction was loaded as an input control. Ponceau S-stained bands (PonS) indicated by asterisks show the amount and quality of the GST fusion proteins used in this assay

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The OsNF-YB10 protein could be produced in the GFP-tagged form. Thus, experiments were carried out to investigate the protein–protein interactions between five OsNF-YC proteins tagged with GST and three OsNF-YB proteins tagged with GFP. The GFP-tagged OsNF-YB10 protein interacted with four of the OsNF-YC proteins (OsNF-YC2, OsNF-YC4, OsNF-YC6 and OsNF-YC7), but not with OsNF-YC1 (Fig. 1b). The interactions of GFP-tagged OsNF-YB10 with OsNF-YC6 and OsNF-YC7 proteins were more significant than those with OsNF-YC2 and OsNF-YC4 proteins. This result is similar to the interaction data of the untagged OsNF-YB8 and OsNF-YB11 with OsNF-YC proteins. The GFP-tagged OsNF-YB11 proteins, however, showed somewhat different interaction profiles from those of the untagged proteins. The main difference was that the tagged OsNF-YB11 proteins did not show significant interaction with OsNF-YC1 and OsNF-YC4 proteins. The tagged OsNF-YB11 protein interacted with only three OsNF-YC proteins (OsNF-YC2, OsNF-YC6 and OsNF-YC7) but their interactions were much weaker than the input control. The tagged OsNF-YB8 protein interacted with all five OsNF-YC proteins, but the interaction with the OsNF-YC1 protein was much weaker than with the other four proteins. In general, the tagged OsNF-YB11 proteins showed weaker interactions than the untagged proteins. These data indicate that three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11) could physically interact in vitro with at least three OsNF-YC proteins (OsNF-YC2, OsNF-YC6 and OsNF-YC7).

Since NF-Y transcription factors are known to regulate the expression of diverse genes via direct binding to CCAAT motifs within the promoter regions (Frontini et al. 2004; Steidl et al. 2004; Goda et al. 2005; Tuncher et al. 2005), subcellular localization experiments in tobacco leaves were also carried out to investigate whether OsNF-YB and OsNF-YC proteins are nuclear proteins. The OsNF-YB11-GFP protein was present in both the nucleus and the cytoplasm, whereas the OsNF-YB8-GFP protein was found only in the cytoplasm (Supplementary Fig. S1). The OsNF-YB10-GFP protein, however, occurred only in the nucleus. The five OsNF-YC-GFP proteins were localized both in the nucleus and the cytoplasm (Supplementary Fig. S1). This indicates that the subcellular localization patterns of the OsNF-YB and OsNF-YC proteins overlap in planta.

The overlapping localization patterns of the OsNF-YB-GFP proteins with the OsNF-YC-GFP proteins suggest the possibility that the OsNF-YB proteins may interact with the OsNF-YC proteins in vivo. A BiFC assay in Agrobacterium-infiltrated tobacco leaves therefore was performed using N- or C-terminal yellow fluorescent protein (YFP) fragment fused OsNF-YBs and OsNF-YCs constructs (Walter et al. 2004). YFP signals were observed in the nucleus and cytoplasm of the transformed cells with all combinations of OsNF-YB and OsNF-YC proteins, indicating that these all interact in planta (Supplementary Fig. S2). Taken together with the in vitro protein–protein interaction data (Fig. 1), these data suggest that OsNF-YB8, OsNF-YB10 and OsNF-YB11 proteins interact with OsNF-YC2, OsNF-YC4 and OsNF-YC6 proteins in planta. Based on this protein–protein interaction data, we decided to characterize the function of only three proteins, OsNF-YC2, OsNF-YC4 and OsNF-YC6 proteins using transgenic approaches.

OsNF-YC proteins are LD-specific flowering regulators

To investigate the effects of the decreased OsNF-YC2, OsNF-YC4 or OsNF-YC6 expression on flowering time in rice, we generated transgenic rice in which the expression of these genes was suppressed by RNA-mediated interference (RNAi). T1 transgenic plants for each construct were obtained and the flowering time of transgenic plants was examined in the T2 generation. In this experiment, T2 lines for the transgenics and their segregated non-transgenic siblings were grown under SD conditions (10 h of light/14 h dark) or LD conditions (14.5 h of light/9.5 h of dark). The endogenous transcript levels of OsNF-YC2, OsNF-YC4 or OsNF-YC6 genes in OsNF-YC2-RNAi, OsNF-YC4-RNAi or OsNF-YC6-RNAi, respectively, decreased to less than 20 % of the corresponding non-transgenic lines (Fig. 2a). However, the transcript levels of other similar OsNF-YC genes were not altered in each transgenic lines, i.e., the expression levels of OsNF-YC1 that occur in the same clade with OsNF-YC4 were very similar in transgenic or non-transgenic OsNF-YC4-RNAi lines (Supplementary Fig. S3). The respective transgenic RNAi plants showed that the flowering phenotypes changed only under LD conditions (Table 1). For statistical significance, we compared the flowering times of transgenic and non-transgenic segregants for each gene. The flowering time of the transgenic OsNF-YC2-RNAi (#7) plants was earlier than that of the non-transgenic lines. The differences of days to heading (DTH) of #7 were  11.4 and 7.5 days earlier than non-transgenic lines in two biological replicate experiments, respectively (Table 1; Fig. 2b). Transgenic OsNF-YC4-RNAi (#100 and #136) plants also exhibited earlier flowering time, compared to the non-transgenic lines (difference of DTH of #100 and #136 = 10 and 5.4 days, respectively), but this result was not replicated in the second biological replicate experiment (Table 1; Fig. 2c). Meanwhile, the flowering time of OsNF-YC6-RNAi (#33) plants was not altered under the same conditions (Table 1; Fig. 2d). These data suggest that the decreased expression of OsNF-YC2 leads to early flowering time only under LD conditions.

Fig. 2
figure 2

Flowering phenotypes of OsNF-YC2-RNAi, OsNF-YC4-RNAi and OsNF-YC6-RNAi transgenic plants in the T1 generation. a The expression levels of endogenous OsNF-YC2, OsNF-YC4 and OsNF-YC6 genes in the segregating lines of the respective transgenic plants. The expression levels of OsUBQ gene were used as a normalization control. Error bars indicate the standard deviation of the three technical replicates. OsNF-YC2-RNAi (b), OsNF-YC4-RNAi (c) and OsNF-YC6-RNAi (d) transgenic plants (+), and their segregating non-transgenic controls (−) grown under long-day (LD) conditions were photographed after the heading of the transgenic plants. Arrows indicate panicles showing heading. Scale bars 10 cm

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Table 1 Flowering time of OsNF-YC2-RNAi, OsNF-YC4-RNAi and OsNF-YC6-RNAi plants
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To further confirm the functional roles of OsNF-YC2, OsNF-YC4 and OsNF-YC6 in terms of flowering time, we made transgenic plants that overexpressed OsNF-YC2, OsNF-YC4 or OsNF-YC6 under the maize ubiquitin1 (Ubi1) promoter. T0 transgenic plants for each construct were obtained and transgenic lines were selected for analysis of flowering time in the T1 generation. The overexpression of OsNF-YC2, OsNF-YC4 or OsNF-YC6 transcripts was observed in the respective transgenic lines (Fig. 3a). Western blot analysis further confirmed that GFP-tagged OsNF-YC2, OsNF-YC4 or OsNF-YC6 proteins were highly expressed in the same transgenic lines (Fig. 3b). Altered flowering phenotypes in the respective transgenic plants were observed only under LD conditions. Transgenic Ubi1::OsNF-YC2:GFP plants (#2, #3, #4 and #5) exhibited severely delayed flowering phenotypes, compared to that of the segregated non-transgenic siblings (difference of DTH = more than 80 days) (Table 2; Fig. 3c). In addition, the transgenic line of Ubi1::OsNF-YC4:GFP (#16 and #19) plants also showed a delay in flowering phenotypes (difference of DTH = more than 65 days) (Table 2; Fig. 3d). In contrast, the transgenic Ubi1::OsNF-YC6:GFP plants (#2) showed an early flowering phenotype, compared to those of segregated nontransgenic siblings and wild-type rice plants (difference of DTH = more than 7 days) (Table 2; Fig. 3e). The difference of DTH among transgenic lines also appeared to correlate well with the degree of expression of the transgenes (Table 2; Fig. 3a, b). The flowering phenotypes of some selected transgenic lines (#1 in Ubi1::OsNF-YC2:GFP, #9 and #16 in Ubi1::OsNF-YC4:GFP, and #2 in Ubi1::OsNF-YC6:GFP) were further confirmed in the T2 generation (Table 2), although the flowering times of segregated nontransgenic T2 siblings were rather divergent from that of wild-type rice plants. This difference may result from the small number of nontransgenic siblings. In addition, we presented RNA and protein expression data of lines #2 and #6 of Ubi1::OsNF-YC6:GFP transgenic plants in the T1 generation (Fig. 3a, b). The flowering times of the two different lines (#2 and #6) correlated with the RNA and protein expression levels. The RNA levels were examined using RT-qPCR analysis which was indicated in the legend of Fig. 3. The difference in flowering time of wild-type rice plants in experiments 2 and 3 was about 17 days. This was not such an extreme difference for the control value because flowering time under LD condition (14.5 h light) was usually more than 110 days in the growth cabinets we have used. These data, taken together with the transgenic approach using RNAi, suggest that OsNF-YC2 and OsNF-YC4 proteins act as flowering repressors and that the OsNF-YC6 protein may function as a flowering promoter only under LD conditions in rice.

Fig. 3
figure 3

Flowering phenotypes of Ubi1::OsNF-YC2:GFP, Ubi1::OsNF-YC4:GFP and Ubi1::OsNF-YC6:GFP transgenic plants in the T1 generation. a The mRNA levels of OsNF-YC2, OsNF-YC4 and OsNF-YC6 genes in the segregating lines of the respective transgenic plants. RT-qPCR was performed to examine the expression levels of the OsNF-YC genes. The expression levels of OsUBQ gene were used as a normalization control. Error bars indicate the standard deviation of the three technical replicates. b Protein levels in the segregating lines of the respective transgenic plants. Anti-GFP antibody (α-GFP) was used for western blot analysis. Ponceau S-stained Rubisco large subunit (rbcL) was used as a loading control. Ubi1::OsNF-YC2:GFP (c), Ubi1::OsNF-YC4:GFP (d) and Ubi1::OsNF-YC6:GFP (e) transgenic plants (+), and their segregating non-transgenic controls (−) grown under LD conditions were photographed after the heading of transgenic or non-transgenic plants. Arrows indicate panicles showing heading. Scale bars 10 cm

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Table 2 Flowering time of Ubi1::OsNF-YC2:GFP, Ubi1::OsNF-YC4:GFP and Ubi1::OsNF-YC6:GFP transgenic plants
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Flowering time genes are affected by strong expression of OsNF-YC2, OsNF-YC4 or OsNF-YC6 genes in transgenic plants

To elucidate the molecular mechanisms by which OsNF-YC2, OsNF-YC4 or OsNF-YC6 regulate flowering time in rice, we investigated the expression of some flowering regulators such as Early heading date 1 (Ehd1) in segregated lines of the respective transgenic plants at T1 and T2 generation (Tables 1, 2). Among the RNAi lines, only OsNF-YC2-RNAi plants showed significant acceleration in flowering time compared to non-transgenic controls, however, change in the expression of flowering time genes responsible for these phenotypes was very subtle (data not shown). Some overexpression lines of OsNF-YC2, OsNF-YC4 or OsNF-YC6 were examined for the expression of flowering time genes by RT-PCR analysis (Supplementary Fig. S4). Ehd1, FTL, Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) expression levels were noticeably changed. Then, transgenic plants showing strong expression of OsNF-YC2, OsNF-YC4 or OsNF-YC6 were further examined for the expression of flowering regulators using RT-qPCR analysis (Fig. 4; Supplementary Figs. S5, S6). In transgenic Ubi1::OsNF-YC2:GFP (#3 and 1–4) and Ubi1::OsNF-YC4:GFP (#19 and 16–12) plants grown under LD conditions, Ehd1, Hd3a, and RFT1 expression decreased (Fig. 4a, b), whereas the expression of these increased in transgenic Ubi1::OsNF-YC6:GFP (#2–1) plants under the same conditions (Fig. 4c). However, the expression level of Ghd7 (Doi et al. 2004; Itoh et al. 2010) increased significantly only in Ubi1::OsNF-YC2:GFP plants, suggesting that the OsNF-YC2 protein may also promote the expression of the Ghd7 gene, which then reduces the expression of Ehd1, Hd3a, and RFT1s under LD conditions (Fig. 4a). The expression levels of OsLFL1 as negative regulators of Ehd1 (Peng et al. 2007) and Hd1 as a negative regulator of Hd3a/RFT1 (Yano et al. 2000; Hayama et al. 2003) under LD conditions were not altered significantly in Ubi1::OsNF-YC2:GFP, Ubi1::OsNF-YC4:GFP, and Ubi1::OsNF-YC6:GFP plants (Supplementary Figs. S4, S5, S6). Other flowering regulators, for example OsMADS50, were also not altered (Supplementary Figs. S4, S5, S6) (Komiya et al. 2009; Ryu et al. 2009). Thus, these data suggest that OsNF-YC4 and OsNF-YC6 proteins affect Ehd1, Hd3a, and RFT1 expression under LD conditions, independently of Ghd7 and Hd1, whereas OsNF-YC2 protein represses the expression of Ehd1, Hd3a, and RFT1 via Ghd7 or the expression of Ehd1, Hd3a, and RFT1 genes directly. Taken together, these data suggest that these three OsNF-YC genes function upstream of Ehd1, Hd3a, and RFT1 as LD-specific regulators.

Fig. 4
figure 4

Hd3a, RFT1, Ehd1 and Ghd7 expression in Ubi1::OsNF-YC2:GFP, Ubi1::OsNF-YC4:GFP, and Ubi1::OsNF-YC6:GFP transgenic plants. Leaves of Ubi1::OsNF-YC2:GFP (a), Ubi1::OsNF-YC4:GFP (b) and Ubi1::OsNF-YC6:GFP (c) transgenic segregants grown under LD conditions were harvested at the initiation of light phase at 41 or 51 days. Two biological replicates were done. RT-qPCR was performed to examine the relative expression levels of the OsNF-YC genes and the flowering time genes. The expression levels were normalized with that of OsUBQ, respectively. The highest level was set at 1. Transgenic plants (+) and their segregating non-transgenic controls (−) were indicated

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The expression patterns of OsNF-YB and OsNF-YC genes

The diurnal oscillation patterns of the subsets of OsNF-YB and OsNF-YC genes, OsCO3, Hd1, and Ghd7 were compared in rice seedlings grown under LD conditions to examine if there was correlation in the expression patterns. OsNF-YB11 and OsNF-YC4 expression showed oscillation patterns with a peak around the end of the light period, which was similar to the pattern observed for Hd1 expression (Fig. 5a). In contrast, the expression levels of OsNF-YC2, Ghd7, and OsCO3 exhibited oscillation patterns with a peak at the beginning of the light period (Fig. 5b). However, the transcripts of OsNF-YB8, OsNF-YB10, and OsNF-YC6 did not show the diurnal oscillation patterns that were observed in OsCO3, Hd1, and Ghd7 (Fig. 5c). These data indicate that specific subsets of OsNF-YB and OsNF-YC genes have similar diurnal oscillation patterns to OsCO3, Hd1, and Ghd7 genes.

Fig. 5
figure 5

Diurnal oscillation patterns of OsNF-YB8, OsNF-YB10, OsNF-YB11, OsNF-YC2, OsNF-YC4, OsNF-YC6, Hd1, Ghd7, and OsCO3. a Similar oscillation patterns among OsNF-YC4, OsNF-YB11, and Hd1. Rice seedlings were harvested every 4 h for 44 h under LD conditions. The expression levels of OsUBQ were used as a normalization control. Error bars indicate standard deviation of the three technical replicates. ZT denotes zeitgeber time. b Similar oscillation patterns among OsNF-YC2, OsCO3, and Ghd7. c Dissimilar patterns of OsNF-YC6, OsNF-YB10, and OsNF-YB8

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The spatial expression patterns of some OsNF-YB, OsNF-YC, and CCT domain-containing genes were also compared in a variety of rice tissues. OsNF-YB11 and OsNF-YC4 and Hd1 expression showed similar expression patterns (Fig. 6a). Abundant expression was observed in leaf sheaths, leaf blades, flag leaves, and roots. OsNF-YC2, Ghd7, and OsCO3 was mainly expressed in the leaf blades and flag leaves (Fig. 6b). This indicates that tissue-specific expression patterns of some OsNF-YB and OsNF-YC genes are similar to those of OsCO3, Hd1, and Ghd7 genes. However, the expression levels of OsNF-YB8, OsNF-YB10, and OsNF-YC6 were not similar to those of OsCO3, Hd1, and Ghd7 (Fig. 6c). Taken together, these data suggest that OsCO3, Hd1 and Ghd7 proteins may interact with specific subsets of OsNF-YB and OsNF-YC proteins, respectively, in the regulation of photoperiodic flowering in rice.

Fig. 6
figure 6

Spatial expression patterns of OsNF-YB8, OsNF-YB10, OsNF-YB11, OsNF-YC2, OsNF-YC4, OsNF-YC6, Hd1, Ghd7, and OsCO3. a Similar expression patterns among OsNF-YC4, OsNF-YB11, and Hd1. Respective samples [culms (C), leaf sheaths (S), leaf blades (B), flag leaves (F), panicles (P), and roots (R)] were harvested from adult rice plants. The expression levels of OsUBQ were used as a normalization control. Error bars indicate standard deviation of three technical replicates. b Similar expression patterns among OsNF-YC2, OsCO3, and Ghd7. c Dissimilar expression patterns of OsNF-YC6, OsNF-YB10, and OsNF-YB8

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Discussion

OsNF-YC proteins form heterodimeric complexes with OsNF-YB proteins

The rice genome contains multiple gene families of OsNF-Y genes (10 OsNF-YA genes, 11 OsNF-YB genes and 7 OsNF-YC genes) (Petroni et al. 2012; Laloum et al. 2013). Overlapping expression patterns of OsNF-YA, OsNF-YB, and OsNF-YC genes have been reported (Thirumurugan et al. 2008). Recently, it has been reported that the OsNF-YB11 (OsHAP3H/Ghd8/DTH8) gene is involved in the regulation of flowering time and other traits such as grain productivity and plant height (Wei et al. 2010; Yan et al. 2011). Thus, an important question is raised as to whether the OsNF-Y subunit interacts with specific members of the other two OsNF-Y subunits to influence flowering time. Several lines of evidence in this study suggest that three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) specifically interact with three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11) in the regulation of flowering time. First, specific protein–protein interactions between three OsNF-YC and three OsNF-YB proteins were observed in vitro and in planta (Fig. 1; Fig. S2). Second, three OsNF-YC proteins were co-localized in the nucleus with three OsNF-YB proteins (Figs. S1, S2). Third, the diurnal oscillation expression and tissue-specific expression patterns of OsNF-YC2 and OsNF-YC4 genes were similar to those of OsNF-YB11, Hd1, Ghd7, and OsCO3 genes (Figs. 5, 6). Finally, in the RNAi suppression or overexpression lines of three OsNF-YC genes, it was shown that the flowering time in rice was affected (Figs. 2, 3; Tables 1, 2). Our conclusion is supported by three OsNF-YC and three OsNF-YB proteins that are clustered in the same phylogenetic clade (Thirumurugan et al. 2008; Petroni et al. 2012; Laloum et al. 2013) as AtNF-YC3/-YC4/-YC9 proteins and AtNF-YB2/-YB3 proteins, which are known to influence flowering (Kumimoto et al. 2008, 2010). However, the possibility that the OsNF-YC subunits interact with other OsNF-YB subunits in other developmental processes, cannot be excluded. This is based both on our finding that each OsNF-YC subunit interacts with three OsNF-YB proteins (Fig. 1; Figs. S1, S2), the expression of some OsNF-Y genes was detected in all tissues tested in this study (Fig. 6), and the report of Thirumurugan et al. (2008) that OsNF-YB2, which is involved in chloroplast biogenesis (Miyoshi et al. 2003), interacts with OsNF-YC subunits in yeast.

OsNF-YC2, OsNF-YC4, and OsNF-YC6 proteins act as LD-specific flowering regulators in the regulation of the photoperiod-dependent flowering response in rice

Since AtNF-YC3, AtNF-YC4 and AtNF-YC9 proteins regulate the photoperiod-dependent flowering through interaction with CO protein (Kumimoto et al. 2010), we investigated whether three OsNF-YC proteins regulate the photoperiod-dependent flowering response in rice. The present study provides evidence that OsNF-YC2, OsNF-YC4, and potentially or possibly OsNF-YC6 proteins act as LD-specific flowering regulators in rice. First, transgenic approaches using RNAi suppression or overexpression revealed that three OsNF-YC genes modulated flowering time only under LD conditions (Figs. 2, 3; Tables 1, 2). Although OsNF-YC4-RNAi and OsNF-YC6-RNAi plants did not show any significant change in flowering time, OsNF-YC2-RNAi plants did show significant acceleration in flowering time when compared to non-transgenic controls. The lack of phenotypic effect in OsNF-YC4-RNAi and OsNF-YC6-RNAi plants could be due to the functional redundancy of these genes with the homologous OsNF-YC genes (Thirumurugan et al. 2008; Petroni et al. 2012; Laloum et al. 2013). Transgenic plants over-expressing the OsNF-YC genes showed either delayed or accelerated flowering only under LD conditions (Fig. 3; Table 2). Second, the overexpression of three OsNF-YC genes affected the expression levels of Ehd1, Hd3a, and RFT1 acting within a LD-specific pathway (Komiya et al. 2009) (Fig. 4). These findings indicate that three OsNF-YC genes primarily regulate the photoperiod-dependent flowering response under LD conditions in rice.

In this study, it has been demonstrated that three OsNF-YC proteins interact with the same OsNF-YB proteins (Fig. 1; Fig. S2). Also, the effects of OsNF-YC2 and OsNF-YC4 on flowering time were the opposite of OsNF-YC6 flowering time (Figs. 2, 3; Tables 1, 2), although the acceleration of flowering by the overexpression of OsNF-YC6 needs further validation. This could be explained as follows: in vivo, OsNF-YB and OsNF-YC heterodimeric complexes may interact with flowering repressor proteins such as Hd1 or Ghd7, or alternatively these may interact with flowering promoter proteins and perform the opposite function. However, the possibility cannot be excluded that OsNF-YC6 affects flowering time via a dominant negative effect when it is ectopically overexpressed, because overexpressed OsNF-YC6 proteins could promote flowering by interrupting the formation of functional complexes. Further investigation on these possibilities would provide a better understanding of protein–protein interactions between OsNF-YB and OsNF-YC proteins.

Possible interactions between OsNF-YB or OsNF-YC proteins, and flowering regulators with a CCT domain in rice

CO protein is known to interact with AtNF-YC3, 4 and 9 proteins via the CCT domain (Wenkel et al. 2006; Kumimoto et al. 2010). In wheat, VRN2 (acting as a flowering repressor, Yan et al. 2004) and CO2 [acting as a flowering promoter (Nemoto et al. 2003)] also physically interact with the same subset of eight TaNF-Y proteins (Li et al. 2011). In this study, we found a similar subcellular localization of OsNF-YB, OsNF-YC, Hd1, Ghd7 and OsCO3 (Fig. S1), and in vitro and in planta interaction between OsNF-YB and OsNF-YC (Fig. S2). In addition, some OsNF-YB and OsNF-YC genes have similar diurnal and spatial expression patterns with Hd1, Ghd7 and OsCO3 genes (Figs. 5, 6). This suggests that specific subsets of OsNF-YB and OsNF-YC proteins regulate the photoperiodic flowering in rice through direct interaction with flowering regulators with the CCT domain. However, the full-length Hd1, Ghd7 and OsCO3 (functioning as CCT domain proteins) did not physically interact with subsets of either OsNF-YB or OsNF-YC proteins in vitro (data not shown). This suggests that Hd1, Ghd7 and OsCO3 may need appropriate conformational change for the interaction with OsNF-YB or OsNF-YC proteins in vivo, as suggested in a report that the full-length CO does not interact with AtNF-YB2 (Kumimoto et al. 2010). However, we cannot dismiss the possibility that some unknown factors or phosphorylated form of the CCT domain proteins may be required for formation of the heterotrimeric complex among the CCT domain proteins, OsNF-YB and OsNF-YC proteins in vivo. This is supported by evidence that a casein kinase I (Hd16) controls flowering time in rice through the phosphorylation of Ghd7 (Hori et al. 2013; Kwon et al. 2015). Finally, the rice flowering regulators with the CCT domain might bind to only complete OsNF-Y heterotrimeric complexes (OsNF-YA, OsNF-YB and OsNF-YC) to promote flowering in rice. This is supported by the proposal of Cao et al. (2014) that the canonical AtNF-Y complex binds the FT distal enhancer and recruits CO to proximal FT promoter elements. Further investigation will be needed to identify the OsNF-Y protein complexes in vivo.

Author contribution statement

SKK designed research, conducted experiments, analyzed data and wrote the manuscript. HYP, YHJ, and KCL conducted experiments and analyzed data. YSC analyzed data. JHL analyzed data and wrote the manuscript. JKK conceived and designed research, analyzed data and wrote the manuscript. All authors read and approved the manuscript.