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. 2020 Aug 28;19(2):300–310. doi: 10.1111/pbi.13462

OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression

Juntao Chai 1,*, Shanshan Zhu 2,*, Chaonan Li 2,*, Chunming Wang 1, Maohong Cai 1, Xiaoming Zheng 2, Liang Zhou 1, Huan Zhang 1, Peike Sheng 2, Mingming Wu 1, Xin Jin 2, Zhijun Cheng 2, Xin Zhang 2, Cailin Lei 2, Yulong Ren 2, Qibing Lin 2, Shirong Zhou 1, Xiuping Guo 2, Jie Wang 2, Zhichao Zhao 2, Jianmin Wan 1,2,✉
PMCID: PMC7868965  PMID: 32757315

Summary

Heading date is a key agronomic trait affecting crop yield. In rice, Early heading date 1 (Ehd1) is an important B‐type response regulator in determination of heading date. Although many regulatory factors of Ehd1 expression have been functionally characterized, the direct regulators of Ehd1 largely remain to be identified. Here, we identified a new regulator of Ehd1, OsRE1, that directly binds to the A‐box motif in the Ehd1 promoter. Osre1 confers an early heading phenotype due to elevated expression levels of Ehd1. OsRE1 is a nucleus‐localized bZIP transcription factor with a diurnal rhythmic expression pattern. Furthermore, we identified an OsRE1‐interacting protein, OsRIP1, and demonstrated that OsRIP1 can repress the transcript expression of Ehd1 in an OsRE1‐dependent manner. Our genetic data showed that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our results suggest that OsRE1 functions cooperatively with OsRIP1 to regulate heading date through finely modulating the expression of Ehd1. In addition, OsRE1 and OsRIP1 are two minor heading date regulators, which are more desirable for fine‐tuning heading date to improve rice regional adaptability.

Keywords: rice (Oryza sativa), Ehd1, heading date, OsRE1, OsRIP1

Introduction

Rice (Oryza sativa) is one of the most important cereal crops in the world by serving as a staple food for more than half of the world population (Foley et al., 2011). Heading date (flowering time) is an essential biological trait in rice that influences regional adaptation and grain production (Izawa, 2007). Heading date is determined by multiple external and internal signals, including photoperiod, temperature and hormones (Luan et al., 2009). Photoperiod is the most important environmental factor affecting heading date in rice (Song et al., 2015), a typical short‐day plant. Several flowering pathways affected by photoperiod were identified (Sun et al., 2014). The HEADING DATE1 (Hd1) pathway is conserved both in rice (OsGI‐Hd1‐Hd3a) and in Arabidopsis thaliana (GI‐CO‐FT) (Shrestha et al., 2014). OsGI (GIGANTEA) and Hd1 in rice are homologs of GI and CO (CONSTANS) in Arabidopsis, respectively (Hayama et al., 2002; Yano et al., 2000). Unlike CO which merely promotes flowering in Arabidopsis, Hd1 plays a dual role in rice by promoting flowering under short‐day (SD) conditions and repressing flowering under long‐day (LD) conditions (Hayama et al., 2003; Zhang et al., 2017). The EARLY HEADING DATE1 (Ehd1) pathway is unique to rice and was reported to function independent of OsGI‐Hd1‐Hd3a in controlling heading date (Doi et al., 2004). Both pathways target the florigens HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1), which have high amino acid sequence similarity with FLOWERING LOCUS T (FT), the florigen in Arabidopsis (Kojima et al., 2002; Komiya et al., 2008; Tamaki et al., 2007). Notably, Hd1 and PLANT HEIGHT AND HEADING DATE 7 (Ghd7, a strong flowering repressor in Ehd1 pathway) were reported to form complex, which specifically bind to a cis‐regulatory region in Ehd1 promoter and repressed its expression, suggesting that the OsGI‐Hd1‐Hd3a pathway likely integrates into the Ehd1 pathway under LD conditions (Nemoto et al., 2016).

Ehd1 encodes a B‐type response regulator (Doi et al., 2004). Many proteins regulate Ehd1 expression. OsMADS50, OsMADS51, RICE INDETERMINATE 1 (RID1)/EARLY HEADING DATE 2 (Ehd2), EARLY HEADING DATE 3 (Ehd3), EARLY HEADING DATE 4 (Ehd4), Hd3a BINDING REPRESSOR FACTOR 1 (HBF1) and SDG723/OsTrx1/OsSET33 INTERACTION PROTEIN 1 (SIP1) function as positive regulators of Ehd1 (Brambilla et al., 2017; Gao et al., 2013; Jiang et al., 2018; Kim et al., 2007; Lee et al., 2004; Matsubara et al., 2011; Matsubara et al., 2008; Wu et al., 2008). OsCOL4, GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7), DAYS TO HEADING ON CHROMOSOME 8 (DTH8/Ghd8/OsHAP3H), OsCOL9, OsCOL10, OsCOL13, OsHAPL1, OsMFT1 and OsIDS1 function as repressors of Ehd1 (Lee et al., 2010; Lee et al., 2014; Liu et al., 2016; Sheng et al., 2016; Song et al., 2018; Tan et al., 2016; Wei et al., 2010; Xue et al., 2008; Zhu et al., 2017). Of them, only HBF1 and SIP1 bind to the Ehd1 promoter directly (Brambilla et al., 2017; Jiang et al., 2018). Although many regulators of Ehd1 have been identified, direct regulators are rarely reported, and the mechanisms by which Ehd1 is directly regulated have remained elusive.

The basic leucine (Leu) zipper (bZIP) transcription factor (TF) family is one of the largest and most diverse TF families in plants (Dröge‐Laser et al., 2018). bZIP proteins are named by their conserved bZIP domain, which contains 60‐80 amino acids (Landschulz et al., 1988). There are 89 bZIP‐encoding genes in rice genome (Nijhawan et al., 2008). bZIP proteins participate in multiple biological processes including pathogen defence, response to abiotic stress, phytohormone activity, flowering, senescence, seed development and germination (E et al., 2014). In Arabidopsis, bZIP protein FD interacts with FT in the shoot apical meristem (SAM) to promote flowering (Abe et al., 2005; Wigge et al., 2005). In rice, the homolog of FD, OsFD and an additional bZIP protein HBF1(Hd3a BINDING REPRESSOR FACTOR 1) interact with Hd3a/RFT1 in the SAM and regulate Ehd1 expression in leaves (Brambilla et al., 2017). Another bZIP protein, OsABF1 (ABA RESPONSIVE ELEMENT BINDING FACTOR 1), mediates drought‐derived signals to regulate heading date through indirect suppression of Ehd1 (Zhang et al., 2016).

BBX proteins (B‐box‐containing proteins) are a class of zinc finger proteins containing one or two B‐box domains (Borden, 1998; Torok and Etkin, 2001). 30 BBX genes (OsBBXs) were identified in rice through genome‐wide surveys (Huang et al., 2012). OsBBX proteins are divided into five subfamilies according to the number of B‐box domains and presence or absence of a CCT domain (Griffiths et al., 2003). BBX proteins play important roles in plant growth and development, such as seedling photomorphogenesis, photoperiodic regulation of flowering, shade avoidance and response to biotic or abiotic stress (Cheng and Wang, 2005; Crocco et al., 2015; Gangappa et al., 2013; Kumagai et al., 2008). The BBX proteins Hd1 and OsCO3, known as CO homologs in rice, have been reported to regulate heading date (Kim et al., 2008; Yano et al., 2000). OsBBX14, encoding a BBX TF with two B‐boxes and no CCT motif, delays heading date by acting as an indirect repressor of Ehd1 (Bai et al., 2016). Although BBX protein is reported to affect flowering time, its physiological significance and its regulatory mechanism have remained elusive.

Here, we identified a direct regulator of Ehd1, OsRE1, a bZIP transcription factor that was localized in the nucleus and exhibited rhythmical expression pattern. Osre1 showed earlier heading date with elevated expression levels of Ehd1. Biochemical assays showed that OsRE1 physically interacts with OsRE1‐INTERACTING PROTEIN 1 (OsRIP1), a BBX transcription factor. Osrip1 also shows early heading with elevated expression levels of Ehd1. Biochemical and genetic evidence showed that OsRIP1 cannot directly bind to but facilitate OsRE1 to bind to the Ehd1 promoter to repress the Ehd1 expression. Furthermore, our genetic data suggest that OsRE1 and OsRIP1 may function upstream of Ehd1. Our combined molecular, biochemical and genetic data support this notion that OsRE1 forms a transcriptional inhibition complex with OsRIP1 and these factors act cooperatively in regulating heading date through finely repressing the transcript expression of Ehd1 in rice.

Results

OsRE1 could directly bind to the Ehd1 promoter in vivo and in vitro

To identify direct regulators of Ehd1, yeast one‐hybrid (Y1H) library screening assay was performed using an Ehd1 promoter fragment. LOC_Os01g07880, a bZIP transcription factor, was selected as a potential candidate. We designated it regulator of Ehd1, OsRE1. To further confirm the precise binding region for OsRE1, the Ehd1 promoter was divided into five fragments for Y1H assay. OsRE1 specifically bound to P2, P3 and P5 fragments but not P1 and P4 fragments (Figure 1a). Interestingly, we found that P2, P3 and P5 fragments constantly contain an A‐box motif (TACGTA; 638 bp in the Ehd1 promoter), a preferential binding site for bZIP proteins (Hartmann et al., 2005; Izawa et al., 1994). To confirm the interaction in vivo, we performed a chromatin immunoprecipitation (ChIP)‐qPCR assay using Flag‐OsRE1 transgenic plants. Region S4 (−692 to −537 bp) containing the A‐box motif was clearly enriched in the presence of the fused protein (Figure 1b), indicating that OsRE1 could bind to the Ehd1 promoter in vivo.

Figure 1.

Figure 1

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OsRE1 directly binds to the promoter of Ehd1. (a) Y1H assay showed that OsRE1 can bind to the Ehd1 promoter fragments containing A‐box motif (−638 bp). The promoter of Ehd1 was divided into five fragments (P1 to P5). An empty vector was used as the negative control. Yeast was grown on Synthetic Drop‐Out (SD) medium containing X‐gal and lacking Trp/Ura. (b) ChIP‐qPCR assays showing in vivo binding of OsRE1 to the Ehd1 promoter. Cross‐linked chromatin samples were extracted from Flag‐OsRE1 transgenic plants and then precipitated with anti‐Flag antibody. NoAb (No antibody) served as a negative control. Values are means ± SD; n = 3. Asterisks indicate significantly different values (**, P < 0.01). (c) EMSA showed that OsRE1 binds to the A‐box motif of the Ehd1 promoter. The hot probe was a biotin‐labelled fragment of the Ehd1 promoter containing the A‐box motif, and the cold probe was a nonlabelled competitive probe. The mutant probe was the labelled hot probe sequence with two nucleotides mutated. His‐tagged OsRE1 was purified, and 2 μg protein was used. [Colour figure can be viewed at wileyonlinelibrary.com]

We next performed electrophoretic mobility shift assay (EMSA) using a biotin‐labelled Ehd1 promoter fragment (−656 to −615 bp) containing the A‐box motif (−638 to −633 bp). As shown in Figure 1c, OsRE1 bound to the probe containing the A‐box motif (lane 2) and the shift band was suppressed when a competitor probe was used (lanes 3–5), whereas a labelled probe containing two mutated nucleotides did not bind (lane 6). Collectively, these results indicated that OsRE1 can directly bind to the Ehd1 promoter.

OsRE1 is a minor‐effect negative regulator of Ehd1

To investigate whether OsRE1 is involved in determination of heading date in rice, we generated the Osre1 mutant in rice cultivar Nipponbare using the CRISPR‐Cas9 technology. We selected two independent homozygous transgenic lines with different editing (+1 bp and −2 bp) for further study (Figure 2a). Compared with Nipponbare, the Osre1 mutant exhibited a 3.7‐d early heading phenotype under NLD conditions in Beijing (Figure 2b, c). Agronomic traits including plant height, panicle length and grains per panicle were differentially reduced compared to WT, but 1000‐grain weight is not changed (Table S1). Given that OsRE1 could bind to the Ehd1 promoter, we examined whether the expression level of Ehd1 was affected by OsRE1. As shown in Figure 2d, the expression level of Ehd1 was higher in the Osre1 mutant than in WT under LD conditions. Together, these results suggested that OsRE1 is a minor‐effect negative regulator of Ehd1.

Figure 2.

Figure 2

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Loss of OsRE1 function promotes flowering. (a) Mutation sites in OsRE1 modified by the CRISPR‐Cas9 genome editing. The guide RNA (gRNA) spacer sequence is shown in blue, and the protospacer adjacent motif (PAM) site is shown in red. Deleted nucleotides are depicted as dashes, and inserted nucleotides are shown in green. Nip, Nipponbare. (b) Phenotypes of Nip and Osre1 under NLD conditions. 3# and 7# represent two independent Osre1 mutant lines. White arrows indicate panicles. Nip, Nipponbare. (c) Statistical analysis for heading date of Nip and Osre1; the mutants showing earlier flowering. Values are means ± SD; n > 20. Asterisks indicate significantly different values (**, P < 0.01). Nip, Nipponbare. (d) Rhythmic expression patterns of Ehd1 in Nip and Osre1 under LD conditions. The plants were grown in growth chambers under LD (14‐h light/10‐h darkness) conditions for 50 days. Black and white boxes denote dark and light periods, respectively. Rice UBIQUITIN gene was used as the internal control. Values are means ± SD; n = 3. ZT, zeitgeber time. Nip, Nipponbare. [Colour figure can be viewed at wileyonlinelibrary.com]

Subcellular localization of OsRE1 and expression pattern of OsRE1

To determine the subcellular localization of OsRE1, we fused it with green fluorescent protein (GFP) under the control of the cauliflower mosaic virus CaMV 35S promoter (35S: OsRE1‐GFP) and used mCherry‐fused D53 as a nuclear marker (Zhou et al., 2013). As shown in Figure 3a, OsRE1‐GFP fluorescence signals are well merged with the D53‐mCherry signals, suggesting that OsRE1 is a nuclear protein.

Figure 3.

Figure 3

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Subcellular localization of OsRE1 and expression profiles of OsRE1. (a) Subcellular localization of OsRE1 in rice protoplasts. D53‐mCherry protein was used as a nuclear marker. Scale bar, 5 μm. (b) qRT‐PCR analysis of OsRE1 expression levels in different tissues. R, root; St, stem; Sh, sheath; DL‐1, uppermost leaves; DL‐2, second uppermost leaves; YP, young panicles; OP, old panicles. Values are means ± SD; n = 3. (c) Rhythmic expression of OsRE1 in second uppermost leaves under LD (14‐h light/10‐h darkness) and SD (10‐h light/14‐h darkness) conditions. Rice UBIQUITIN gene was used as the internal control. Values are means ± SD; n = 3. (d) Expression analysis of OsRE1 in second uppermost leaves under continuous light (LL) and continuous darkness (DD) conditions. The plants were grown in growth chambers under LD conditions for 30 days and then transferred to LL or DD conditions for 2 days. Black and white bars indicate darkness and light, respectively. Grey bars represent the subjective darkness in DD and subjective light in LL. Rice UBIQUITIN gene was used as the internal control. Values are means ± SD; n = 3. [Colour figure can be viewed at wileyonlinelibrary.com]

To examine the temporal and spatial expression pattern of OsRE1, qRT‐PCR was performed to analyse the expression levels of OsRE1 in different tissues including root, stem, sheath, leaves and panicles. OsRE1 was expressed in all tissues examined, with a relatively high expression in leaves and panicles (Figure 3b).

We then compared the diurnal expression patterns of OsRE1 under both LD (14‐h light/10‐h darkness) and SD (10‐h light/14‐h darkness) conditions. Similar diurnal expression patterns of OsRE1 were evidently observed under both LD and SD conditions. Expression levels rose quickly to a peak at dawn and then decreased rapidly to the lowest point at dusk (Figure 3c). After Nipponbare grown in LD conditions was transferred to continuous light or darkness, rhythmic expression patterns of OsRE1 were destroyed (Figure 3d).

OsRE1 physically interacts with OsRIP1 in vitro and in vivo

To further investigate the function of OsRE1 in determination of heading date, we performed yeast two‐hybrid (Y2H) screening using pGBKT7‐OsRE1 as bait. We screened a cDNA library prepared from leaves and identified a positive clone, namely OsRE1‐INTERACTING PROTEIN 1 (OsRIP1, LOC_Os04g45690). Blast search showed that OsRIP1 was annotated as the BBX transcription factor, OsBBX13 (Huang et al., 2012), but its function remains unknown. Similar to the typical BBX proteins, OsRIP1 contains two conserved B‐box domains (Gangappa and Botto, 2014; Figure S1, Table S3). We further isolated coding sequence for full‐length OsRIP1 and confirmed its interaction with OsRE1 (Figure 4a). To determine the region of both OsRE1 and OsRIP1 for their interaction, we constructed a deletion series of OsRIP1 based on domain prediction (http://pfam.xfam.org/) and examined the interaction with full‐length OsRE1 and vice versa. We identified the bZIP domain of OsRE1 and the second B‐box domain of OsRIP1 as the necessary domains for the interaction between OsRE1 and OsRIP1 (Figure S2a, b).

Figure 4.

Figure 4

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OsRE1 physically interacts with OsRIP1. (a) Yeast two‐hybrid assay showed that OsRE1 interacts with OsRIP1. Yeast was grown at 30 °C for 3 days. Empty vectors were used as the negative controls. AD, activation domain. BD, binding domain. (b) In vitro pull‐down assay showed that OsRE1‐His pulls down MBP‐OsRIP1 but not free MBP. Free MBP and His resin were used as the negative controls. (c) Bimolecular fluorescence complementation (BiFC) assays showed OsRE1 interacts with OsRIP1 in nucleus (labelled by fluorescence of 4′, 6‐diamino‐2‐phenylindole) in leaf epidermal cells of N. benthamiana. Scale bars, 20 μm. (d) In vivo Co‐IP assay showed that Flag‐OsRIP1 can be co‐immunoprecipitated in the total leaf extract N. benthamiana expressing OsRE1‐GFP but not free GFP using anti‐GFP agarose beads. [Colour figure can be viewed at wileyonlinelibrary.com]

An in vitro pull‐down assay confirmed that MBP‐OsRIP1, but not free MBP, pulled down the OsRE1‐His (Figure 4b). To visualize the interaction between OsRE1 and OsRIP1, we performed a biomolecular fluorescence complementation (BiFC) assay in leaf epidermal cells of N. benthamiana. Similar to OsRE1, OsRIP1 displayed a nucleus‐localized pattern in rice protoplasts (Figure S3a). When OsRE1‐eYNE was coexpressed with eYCE‐OsRIP1, strong eYFP fluorescence signals were observed in the DAPI (4′, 6‐diamino‐2‐phenylindole)‐labelled nucleus (Figure 4c). In addition, a firefly luciferase complementation imaging (LCI) assay confirmed the interaction in leaves of N. benthamiana (Figure S2c). Furthermore, an in vivo co‐immunoprecipitation (Co‐IP) assay showed that Flag‐OsRIP1 could be co‐immunoprecipitated by OsRE1‐GFP but not free GFP in total leaf extract of N. benthamiana (Figure 4d).

qRT‐PCR analyses showed that OsRIP1 displayed a similar diurnal expression pattern with OsRE1 (Figure S3b‐d). To investigate whether OsRIP1 is authentically involved in heading, we generated the Osrip1 mutant using the CRISPR‐Cas9 technology. Two homozygous Osrip1 mutant lines with different editing (−1 bp and +1 bp) were selected for further study (Figure 5a). These lines flowered 8.5 days earlier than Nipponbare when grown under NLD conditions in Beijing (Figure 5b, c). Agronomic traits including plant height, panicle length and grains per panicle were reduced relative to Nipponbare, but 1000‐grain weight is not unaffected (Table S1). Together, these results suggested that OsRIP1 is required for flowering in rice.

Figure 5.

Figure 5

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Loss‐of‐function mutation of OsRIP1 promotes flowering. (a) Mutation sites in OsRIP1 induced by the CRISPR‐Cas9 genome editing system. The guide RNA (gRNA) spacer sequence is shown in blue, and the protospacer adjacent motif (PAM) site is shown in red. Deleted nucleotides are depicted as dashes, and inserted nucleotides are shown in green. (b) Phenotypes of Nip and Osrip1 mutant under NLD conditions; 2# and 4# represent two independent Osrip1 mutant lines. White arrows indicate panicles. Nip, Nipponbare. (c) Statistical data for heading date of Nip and Osrip1 mutant under NLD conditions. Nip, Nipponbare. Values are means ± SD; n > 20. Asterisks indicate significantly different values (**, P < 0.01). (d) Rhythmic expression patterns of Ehd1 in Nip and Osrip1 backgrounds under LD conditions. The plants were grown in growth chambers under LD (14‐h light/10‐h darkness) conditions for 50 days. Black and white boxes denote dark and light periods, respectively. Rice UBIQUITIN gene was used as the internal control. Values are means ± SD; n = 3. ZT, Zeitgeber time. Nip, Nipponbare. [Colour figure can be viewed at wileyonlinelibrary.com]

OsRIP1 works cooperatively with OsRE1 to repress the transcript expression of Ehd1

The expression level of Ehd1 was also higher in the Osrip1 mutant than in WT, suggesting that OsRIP1 participates in the Ehd1 pathway (Figure 5d). To determine whether OsRIP1 directly binds to the Ehd1 promoter, we performed Y1H and the result showed that OsRIP1 could not bind the Ehd1 promoter (Figure S4). An in vitro EMSA and in vivo ChIP assay further confirmed OsRIP1 could not bind the Ehd1 promoter (Figure 6a, b). Given that the physical interaction between OsRIP1 and OsRE1, we proposed that OsRIP1 may function cooperatively with OsRE1 to repress the transcriptional activity of Ehd1. A quantitative transactivation assay was performed to investigate the regulatory relationship of OsRE1 and OsRIP1 in modulating the activity of Ehd1. A 2000 bp fragment upstream from start codon of Ehd1 was fused to LUC as the reporter. OsRE1‐GFP and OsRIP1‐Flag were used as effectors with GFP and 3 × Flag as controls (Figure 6c). Luciferase (LUC) activity was reduced by one‐half when ProEhd1: LUC was co‐transfected with 35S‐OsRE1‐GFP. Strikingly, when both OsRE1 and OsRIP1 proteins were coexpressed, the LUC activity declined to one‐third of the control (Figure 6d), suggesting that OsRIP1 facilitates OsRE1 to repress the transcript expression of Ehd1.

Figure 6.

Figure 6

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OsRE1 functions cooperatively with OsRIP1 to negatively regulate Ehd1 expression. (a) EMSA showed that OsRIP1 cannot directly bind to but facilitate OsRE1 to bind to the A‐box motif of the Ehd1 promoter. The hot probe was a biotin‐labelled A‐box motif‐containing fragment in the Ehd1 promoter (sequence showed in Figure 1c). (b) ChIP‐qPCR assay showed in vivo binding of OsRIP1 to the Ehd1 promoter is dependent on OsRE1. Cross‐linked chromatin samples were extracted from Flag‐OsRIP1 and osre1/Flag‐OsRIP1 transgenic plants and then precipitated with Flag antibody. NoAb (No antibody) served as a negative control. Values are means ± SD; n = 3. (c) Schematic diagram of various constructs used in the transient transformation assay. 35S: REN‐ProEhd1: LUC was constructed as the reporter. 35S: OsRE1‐GFP and 35S:3 × Flag‐OsRIP1 were constructed as effectors. Free GFP and Flag were used as negative controls. (d) In vivo luciferase assay verified the inhibitory effect of OsRE1 and OsRIP1 on Ehd1 expression in N. benthamiana leaves. Expression level of Renilla (REN) was used as an internal control. The LUC/REN ratio represents the relative activity of the Ehd1 promoter. Values are means ± SD; n = 3. Asterisks indicate significantly different values (*, P < 0.05; **, P < 0.01).

Genetic interaction between OsRE1, OsRIP1 and Ehd1

To further investigate the genetic interactions between OsRE1, OsRIP1 and Ehd1, we generated Osre1 ehd1 and Osrip1 ehd1 double mutants. Homozygous Osre1 ehd1 and Osrip1 ehd1 double mutants were selected by PCR‐based genotyping (Figure S5). As shown in Figure 7, both Osre1 ehd1 and Osrip1 ehd1 double mutants show a more similar phenotype with ehd1 than with Nipponbare and each single mutant, suggesting that OsRE1 and OsRIP1 function upstream of Ehd1. Additionally, we also noted the subtle difference between double mutants and ehd1 (Figure 7), suggesting the possible existence of unknown targeted genes of OsRE1 and OsRIP1 in regulating heading date independent of Ehd1. To further investigate the downstream genes regulated by OsRE1 and OsRIP1, qRT‐PCR assays were performed. We found the expression of Hd3a, RFT1, MADS14 and Ghd7 was also up‐regulated in the Osre1 and Osrip1 mutant (Figure S6).

Figure 7.

Figure 7

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Osre1 ehd1 and Osrip1 ehd1 double mutants show a more similar phenotype with ehd1. (a) Phenotypes of Nip, Osre1, ehd1 and Osre1 ehd1 at heading stage under NLD conditions. Nip, Nipponbare. (b) Statistical analyses of days to heading of Nip, Osre1, ehd1 and Osre1 ehd1 plants under NLD conditions. Nip, Nipponbare. Values are means ± SD; n > 20. NLD, natural long days. (c) Phenotypes of Nip, Osrip1, ehd1 and Osrip1 ehd1 at the heading stage under NLD conditions. Nip, Nipponbare. (d) Statistical analyses of days to heading of Nip, Osrip1, ehd1 and Osrip1 ehd1 under NLD conditions. Nip, Nipponbare. Values are means ± SD; n > 20. NLD, natural long days. [Colour figure can be viewed at wileyonlinelibrary.com]

Discussion

Although previous studies showed many heading date genes were cloned, most of them are major genes, hard to be utilized in rice breeding. Minor‐effect genes regulate rice heading date weakly, which can regulate rice flowering at the right time, and let rice make full use of the conditions of light and temperature, so these genes can be used for rice heading date breed improvement. Here, we identified OsRE1 as a direct regulator of Ehd1. OsRE1 was annotated as OsbZIP1 in past study, inducible in response to infection with Magnaporthe grisea (Meng et al., 2005). In our study, we found OsRE1 participated in the rice heading date pathway. Furthermore, we found that OsRIP1, a BBX‐like transcription factor, interacts with OsRE1 in vitro and in vivo. Both proteins were located in the nucleus and exhibited similar rhythmical expressions. Loss‐of‐function mutations of OsRE1 and OsRIP1 promote heading date in rice. Our genetic data suggested that OsRE1 and OsRIP1 may function upstream of Ehd1 in regulating heading date. Together, our combined data suggested that OsRE1 and OsRIP1 act as two minor‐effect negative regulators of heading date in rice.

OsRE1 represses Ehd1 expression by directly binding to its promoter

Ehd1 is the key gene in a unique heading date pathway in rice. Although many genes participate in the Ehd1 pathway, direct regulators of Ehd1 largely remain to be identified. Previous studies showed that OsLFL1, a putative B3 transcription factor, binds to RY cis‐elements (CATGCATG) in the Ehd1 promoter (Peng et al., 2007). OsLFL1 functions downstream of OsMADS50 and OsMADS56 to repress the expression of Ehd1 (Ryu et al., 2009). In this study, our EMSA and ChIP assay showed that OsRE1 can directly bind to the Ehd1 promoter (Figure 1). Furthermore, an A‐box motif 638 bp upstream of the ATG of Ehd1 was determined to be the binding site of OsRE1 (Figure 1). The binding site of OsLFL1 (−883 bp and −557 bp) is close to that of OsRE1 (−638 bp). The ACGT motif is also a potential binding site for B3 proteins (Suzuki et al., 1997). Both OsLFL1 and OsRE1 can repress the expression of Ehd1. It will be an interesting avenue to determine whether OsRE1 functions cooperatively with or independently of OsLFL1 in repressing the Ehd1 expression. The C2H2 zinc finger protein SIP1 is another direct regulator of Ehd1 (Jiang et al., 2018). SIP1 participates in regulation of heading date in rice by recruiting OsTrx1 to Ehd1. We found that OsRE1 cannot interact with SIP1 in yeast (data not shown), suggesting that OsRE1 may not be involved in the pathway of SIP1‐mediated Ehd1 regulation.

Recent studies showed that Hd1 also represses expression of Ehd1 through interaction with Ghd7 (Nemoto et al., 2016), but whether Hd1 and Ghd7 could bind to the Ehd1 promoter directly was not clear. DTH8 interacts with Hd1 to repress Hd3a expression by changing the H3K27me3 level in the Hd3a promoter (Du et al., 2017). Ghd7 can also interact with DTH8. It was reported that Ghd7, Hd1 and DTH8 can form a protein complex (Cai et al., 2019). Whether the Ghd7‐Hd1‐DTH8 complex interacts with the Ehd1 promoter is yet to be established.

DTH8 was reported to down‐regulate expression of Ehd1 indirectly (Wei et al., 2010), but the detailed mechanism was not clear. In this study, OsRE1 directly interacts with DTH8 but not with Ghd7 or Hd1 (Figure S7a, b), and OsRE1 represses expression of Ehd1 (Figure 2d). The expression level of OsRE1 was upgraded in the dth8 mutant, while it was not changed in ghd7 mutant (Figure S7c). Therefore, OsRE1 might function as a bridge between DTH8 and Ehd1 in regulating heading date in rice, as the reported DTH8‐Hd1 module (Du et al., 2017; Zhu et al., 2017).

OsRIP1 delays heading date in an OsRE1‐dependent manner

OsRIP1 was previously annotated as OsBBX13 (Huang et al., 2012), but the exact function remains unknown. BBX proteins have been reported to play important roles in plant growth and development, including photoperiodic regulation of flowering (Cheng and Wang, 2005; Crocco et al., 2015; Gangappa et al., 2013; Kumagai et al., 2008). Here, we found that OsRIP1 regulates rice heading date by repressing Ehd1 expression in an OsRE1‐dependent manner. Both Osre1 and Osrip1 mutants showed the early heading phenotype than wild‐type Nipponbare (Figures 2; 5). The Osre1 ehd1 and Osrip1 ehd1 double mutants flowered much later than Nipponbare, Osre1 and Osrip1 (Figure 7). These findings suggested that OsRE1 and OsRIP1 genetically function upstream of Ehd1. Consistently, genes downstream of Ehd1, such as Hd3a, RFT1 and OsMADS14, were promoted in the loss‐of‐function mutants while genes regulating Ehd1, such as DTH8 and Ehd3, were unaffected obviously in the transgenic plants (Figure S6).

Together, our study identified OsRE1 and OsRIP1 as two minor‐effect genes in regulating heading date and thus provides the putative application in crop adaptation and genetic breeding.

Materials and Methods

Plant materials and growth conditions

Transgenic plants were produced using the Oryza sativa subsp. japonica varieties, Nipponbare. All plants were grown in the experimental field at Beijing (40°13′N, 116°34′E) under natural long‐day (NLD) conditions. Plants were also grown in light incubators with constant 70% relative humidity and about 800 μmol/m2/s light intensity under artificial LD (14‐h light, 30°C/10‐h dark, 25°C) or SD (10‐h light, 30°C/14‐h dark, 25°C) conditions.

Generation of transgenic plants

To generate Osre1 and Osrip1 mutants, 18 bp gene‐specific spacer sequences for target genes (Table S2) were inserted into the entry vector pOs‐sgRNA, respectively, followed by subcloning into the destination vector of Cas9 by using the Gateway LR Clonase II Enzyme mix (Invitrogen; Miao et al., 2013). Transgenic plants were PCR‐genotyped, and insertion/deletion mutants at the target site were isolated by DNA sequencing. The primer sequences for construction of these vectors are provided in Table S2.

Yeast one‐hybrid assay

For yeast one‐hybrid assay, the coding sequence (CDS) of OsRE1 was amplified by PCR and cloned into the pB42AD vector (Clontech). To generate ProEhd1: LacZ reporter gene, several fragments of Ehd1 promoter were amplified and cloned into the vector pLacZi. Plasmids were co‐transformed into yeast strain EGY48. Transformants were selected and grown on SD/‐Trp‐Ura dropout medium for 72 h, and then transferred onto X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galacto‐pyranoside) plates for blue colour development (Lin et al., 2007).

Chromatin immunoprecipitation (ChIP)‐qPCR

ChIP assay was performed as previously described (Zhang et al., 2012). Uppermost leaves of Pro35S:3 × Flag‐OsRE1 transgenic plants were used for ChIP assay. Flag antibodies (Medical Biological Laboratories, PM020) were used for detection. The precipitated DNA was analysed by RT‐qPCR using primers listed in Table S2.

Electrophoretic mobility shift assay (EMSA)

The LightShift Chemiluminescent EMSA Kit (Pierce Biotechnology) was used for EMSA. Oligonucleotide probes were synthesized and labelled with biotin by Thermo Fisher Scientific. The OsRE1‐His and MBP‐OsRIP1 fusion proteins were expressed in Escherichia coli strain BL21. The recombinant proteins were purified using either IDA‐Nickel Beads (BEAVER) or amylose resin (BioLabs) affinity chromatography. EMSA was performed according to the manufacturer’s instructions (Thermo, No. 20148). Primer sequences used in EMSA are provided in Table S2.

Subcellular localization

The CDSs of OsRE1 and OsRIP1 were fused at the N‐terminal with GFP in the pAN580 vector, respectively. The GFP fusion plasmids were transformed into rice protoplasts as described previously (Zhang et al., 2011). Pro35S:D53‐mCherry was used as a nucleus marker (Zhou et al., 2013). Fluorescence signals were detected by a ZESS LSM880 confocal microscope.

Yeast two‐hybrid assay

For the yeast two‐hybrid assay, CDS and truncated fragments of OsRE1 and OsRIP1 were amplified and cloned into the pGADT7 or pGBKT7 plasmids (Clontech), respectively. The empty pGADT7 or pGBKT7 vectors were used as negative controls. Yeast co‐transformation was performed following the manual of the Matchmaker Gold Yeast Two‐Hybrid System (Clontech). Different yeast strains were grown on DDO (SD/‐Leu‐Trp) plates and QDO (SD/‐Leu‐Trp‐Ade‐His) plates. The interactions were observed after 3 d of incubation at 30 ℃.

Bimolecular fluorescence complementation (BiFC) assays

The CDSs of OsRE1 and OsRIP1 were fused to YNE or YCE of pDOE‐02 vectors, respectively (Gookin and Assmann, 2014). The plasmids were transformed into Agrobacterium tumefaciens (strain EHA105) and infiltrated into Nicotiana benthamiana leaves as described previously (Waadt and Kudla, 2008). Fluorescence signals were detected by a ZEISS LSM880 confocal microscope.

Pull‐down assays

For the pull‐down assay, the CDS of OsRE1 was fused to the prokaryotic expression vector pET‐30a to express OsRE1‐His. The CDS of OsRIP1 was inserted into pMAL‐c2X to express MBP‐OsRIP1. Plasmids were transformed into E. coli BL21. Fusion proteins were induced with 0.5 mm IPTG at 16 ℃ for 16 h. Pull‐down assays were performed as reported previously (Miernyk and Thelen, 2008). Anti‐His (Medical Biological Laboratories, 1:2000) and anti‐MBP (New England Biolabs, 1:2000) antibodies were used in immunoblotting analysis.

Co‐immunoprecipitation (Co‐IP) assay

The CDS of OsRE1 was fused to pCAMBIA1305.1‐GFP vector. The CDS of OsRIP1 was inserted into pCAMBIA1300‐221‐Flag vector. The plasmids were co‐transformed into Agrobacterium tumefaciens (strain EHA105) and then infiltrated into N. benthamiana leaves. Anti‐GFP and anti‐Flag antibodies (Medical Biological Laboratories, 1:2000) were used in immunoblotting analysis.

Luciferase complementation image (LCI) assay

The CDSs of OsRE1 and OsRIP1 were fused to the pCAMBIA1300‐nLUC and pCAMBIA1300‐cLUC vectors, respectively, for the LCI assay. The empty vectors were used as negative controls. The plasmids were co‐transformed into Agrobacterium tumefaciens (strain EHA105) and then infiltrated into N. benthamiana leaves. CCD imaging apparatus (Berthold, LB985) was used to capture the LUC image. The injected leaves were sprayed with 10 mm beetle luciferin (Promega, E1602) and placed in darkness for 5 min before luminescence detection.

Quantitative transactivation assay

For the quantitative transactivation assay, the 2000 bp promoter of Ehd1 was cloned to fuse into the vector pGreenII0800‐LUC as the reporter. The OsRE1‐GFP and Flag‐OsRIP1 fusion proteins were used as the effectors. Empty vectors were used as negative controls. The above plasmids were co‐transformed into Agrobacterium tumefaciens (strain EHA105) and then infiltrated into N. benthamiana leaves. The Dual‐Glo Luciferase Assay System (E2920, Promega) was used to measure the luciferase (LUC) activity.

Phylogenetic analysis

Gene sequences used in phylogenetic analysis were downloaded and aligned using Geneious (v4.8.5). Alignment data were used to perform maximum‐likelihood analysis. Bootstrap values were estimated from bootstrap analyses of 1000 replicates.

Real‐time PCR assays

Total RNAs of organs were extracted using an RNAprep Pure Kit (Zymo Research, Orange, CA) and were reversely transcribed using a Reverse Transcription Kit (Qiagen). qRT‐PCR was performed using a SYBR Premix Ex Taq II Kit (TaKaRa) in an ABI PRISM 7900HT. Rice UBIQUITIN was used as internal control. Data from three biological replicates were analysed as described previously (Livak and Schmittgen, 2001).

Conflicts of interest

The authors declare no conflict of interest.

Author contributions

Wan Jianmin and Zhu Shanshan designed the research. Chai Juntao, Zhu Shanshan and Li Chaonan performed most of the experiments. Zheng Xiaoming, Zhou Shirong, Cai Maohong and Zhou Liang helped to analysed data. Zhang Huan, Sheng Peike and Wu Mingming helped to generate the double mutants. Cheng Zhijun, Lei Cailin, Wang Jie, Zhao Zhichao and Jin Xin conducted fieldwork. Wang Chunming, Ren Yulong, Lin Qibing, Guo Xiuping and Zhang Xin provided technical support. Chai Juntao, Zhu Shanshan and Li Chaonan contributed equally.

Supporting information

Figure S1 Phylogenetic analysis of BBX family proteins in rice.

Figure S2 OsRE1 physically interacts with OsRIP1.

Figure S3 Expression profiles and subcellular localization of OsRIP1.

Figure S4 OsRIP1 cannot bind to the promoter of Ehd1.in Y1H assay.

Figure S5 Sequencing results of mutant sites in transgenic plants.

Figure S6 Rhythmic expression patterns of genes related to heading date in Nipponbare, Osre1 or Osrip1 under LD conditions.

Figure S7 Interaction relationships between OsRE1, OsRIP1 and several known Ehd1 repressors.

Table S1 Phenotypic data for Nipponbare, Osre1 and Osrip1 mutants.

Table S2 Primers used in this study.

Table S3 Detailed information for rice BBX family members.

Click here for additional data file. (1.6MB, pdf)

Acknowledgements

This work was supported by the National Natural Science Foundation (31771764), the National Key R&D Program of China (2016YFD0100403, 2016YFD0100301 and 2017YFD0100501) and grants from the Fundamental Research Funds for Central Non‐Profit of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (Grant to Zhu Shanshan).

Chai, J. , Zhu, S. , Li, C. , Wang, C. , Cai, M. , Zheng, X. , Zhou, L. , Zhang, H. , Sheng, P. , Wu, M. , Jin, X. , Cheng, Z. , Zhang, X. , Lei, C. , Ren, Y. , Lin, Q. , Zhou, S. , Guo, X. , Wang, J. , Zhao, Z. and Wan, J. (2021) OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression. Plant Biotechnol. J., 10.1111/pbi.13462

References

  1. Abe, M. , Kobayashi, Y. , Yamamoto, S. , Daimon, Y. , Yamaguchi, A. , Ikeda, Y. , Ichinoki, H. et al (2005) FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science, 309, 1052. [DOI] [PubMed] [Google Scholar]
  2. Bai, B. , Zhao, J. , Li, Y. , Zhang, F. , Zhou, J. , Chen, F. and Xie, X. (2016) OsBBX14 delays heading date by repressing florigen gene expression under long and short‐day conditions in rice. Plant Sci. 247, 25–34. [DOI] [PubMed] [Google Scholar]
  3. Borden, K.L.B. (1998) RING fingers and B‐boxes: zinc‐binding protein‐protein interaction domains. Biochem. Cell Biol. 76, 351–358. [DOI] [PubMed] [Google Scholar]
  4. Brambilla, V. , Martignago, D. , Goretti, D. , Cerise, M. , Somssich, M. , de Rosa, M. , Galbiati, F. et al (2017) Antagonistic transcription factor complexes modulate the floral transition in rice. Plant Cell, 29, 2801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai, M. , Chen, S. , Wu, M. , Zheng, T. , Zhou, L. , Li, C. , Zhang, H. et al (2019) Early heading 7 interacts with DTH8, and regulates flowering time in rice. Plant Cell Rep. 38, 521–532. [DOI] [PubMed] [Google Scholar]
  6. Cheng, X.‐F. and Wang, Z.‐Y. (2005) Overexpression of COL9, a CONSTANS‐LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana . Plant J. 43, 758–768. [DOI] [PubMed] [Google Scholar]
  7. Crocco, C.D. , Locascio, A. , Escudero, C.M. , Alabadí, D. , Blázquez, M.A. and Botto, J.F. (2015) The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana . Nat. Commun. 6, 6202. [DOI] [PubMed] [Google Scholar]
  8. Doi, K. , Izawa, T. , Fuse, T. , Yamanouchi, U. , Kubo, T. , Shimatani, Z. , Yano, M. et al (2004) Ehd1, a B‐type response regulator in rice, confers short‐day promotion of flowering and controls FT‐like gene expression independently of Hd1 . Genes Develop. 18, 926–936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dröge‐Laser, W. , Snoek, B.L. , Snel, B. and Weiste, C. (2018) The Arabidopsis bZIP transcription factor family—an update. Curr. Opin. Plant Biol. 45, 36–49. [DOI] [PubMed] [Google Scholar]
  10. Du, A. , Tian, W. , Wei, M. , Yan, W. , He, H. , Zhou, D. , Huang, X. et al (2017) The DTH8‐Hd1 module mediates day‐length‐dependent regulation of rice flowering. Mol. Plant, 10, 948–961. [DOI] [PubMed] [Google Scholar]
  11. E, Z.G. , Zhang, Y.P. , Zhou, J.H. and Wang, L. (2014) Mini review roles of the bZIP gene family in rice. Genetics Mol. Res. 13, 3025–3036. [DOI] [PubMed] [Google Scholar]
  12. Foley, J.A. , Ramankutty, N. , Brauman, K.A. , Cassidy, E.S. , Gerber, J.S. , Johnston, M. , Mueller, N.D. et al (2011) Solutions for a cultivated planet. Nature, 478, 337. [DOI] [PubMed] [Google Scholar]
  13. Gangappa, S.N. and Botto, J.F. (2014) The BBX family of plant transcription factors. Trends Plant Sci. 19, 460–470. [DOI] [PubMed] [Google Scholar]
  14. Gangappa, S.N. , Crocco, C.D. , Johansson, H. , Datta, S. , Hettiarachchi, C. , Holm, M. and Botto, J.F. (2013) The Arabidopsis B‐BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis. Plant Cell, 25, 1243–1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gao, H. , Zheng, X.‐M. , Fei, G. , Chen, J. , Jin, M. , Ren, Y. , Wu, W. et al (2013) Ehd4 encodes a novel and Oryza‐genus‐specific regulator of photoperiodic flowering in rice. PLOS Genet. 9, e1003281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gookin, T.E. and Assmann, S.M. (2014) Significant reduction of BiFC non‐specific assembly facilitates in planta assessment of heterotrimeric G‐protein interactors. Plant J. 80, 553–567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Griffiths, S. , Dunford, R.P. , Coupland, G. and Laurie, D.A. (2003) The evolution of CONSTANS‐like gene families in barley, rice, and Arabidopsis . Plant Physiol. 131, 1855–1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hartmann, U. , Sagasser, M. , Mehrtens, F. , Stracke, R. and Weisshaar, B. (2005) Differential combinatorial interactions of cis‐acting elements recognized by R2R3‐MYB, BZIP, and BHLH factors control light‐responsive and tissue‐specific activation of phenylpropanoid biosynthesis genes. Plant Mol. Biol. 57, 155–171. [DOI] [PubMed] [Google Scholar]
  19. Hayama, R. , Izawa, T. and Shimamoto, K. (2002) Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method. Plant Cell Physiol. 43, 494–504. [DOI] [PubMed] [Google Scholar]
  20. Hayama, R. , Yokoi, S. , Tamaki, S. , Yano, M. and Shimamoto, K. (2003) Adaptation of photoperiodic control pathways produces short‐day flowering in rice. Nature 422, 719–722. [DOI] [PubMed] [Google Scholar]
  21. Huang, J. , Zhao, X. , Weng, X. , Wang, L. and Xie, W. (2012) The rice B‐box zinc finger gene family: genomic identification, characterization, expression profiling and diurnal analysis. PLoS One 7, e48242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Izawa, T. (2007) Adaptation of flowering‐time by natural and artificial selection in Arabidopsis and rice. J. Exp. Botany, 58, 3091–3097. [DOI] [PubMed] [Google Scholar]
  23. Izawa, T. , Foster, R. and Chua, N.H. (1994) Plant bZIP proteins gather at ACGT elements. FASEB J. 8, 192–200. [DOI] [PubMed] [Google Scholar]
  24. Jiang, P. , Wang, S. , Zheng, H. , Li, H. , Zhang, F. , Su, Y. , Xu, Z. et al (2018) SIP1 participates in regulation of flowering time in rice by recruiting OsTrx1 to Ehd1 . New Phytol. 219, 422–435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim, S.L. , Lee, S. , Kim, H.J. , Nam, H.G. and An, G. (2007) OsMADS51 Is a short‐day flowering promoter that functions upstream of Ehd1, OsMADS14 and Hd3a . Plant Physiol. 145, 1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim, S.‐K. , Yun, C.‐H. , Lee, J.H. , Jang, Y.H. , Park, H.‐Y. and Kim, J.‐K. (2008) OsCO3, a CONSTANS‐LIKE gene, controls flowering by negatively regulating the expression of FT‐like genes under SD conditions in rice. Planta 228, 355. [DOI] [PubMed] [Google Scholar]
  27. Kojima, S. , Takahashi, Y. , Kobayashi, Y. , Monna, L. , Sasaki, T. , Araki, T. and Yano, M. (2002) Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short‐day conditions. Plant Cell Physiol. 43, 1096–1105. [DOI] [PubMed] [Google Scholar]
  28. Komiya, R. , Ikegami, A. , Tamaki, S. , Yokoi, S. and Shimamoto, K. (2008) Hd3a and RFT1 are essential for flowering in rice. Development, 135, 767. [DOI] [PubMed] [Google Scholar]
  29. Kumagai, T. , Ito, S. , Nakamichi, N. , Niwa, Y. , Murakami, M. , Yamashino, T. and Mizuno, T. (2008) The common function of a novel subfamily of B‐Box zinc finger proteins with reference to circadian‐associated events in Arabidopsis thaliana . Biosci., Biotechnol. Biochem. 72, 1539–1549. [DOI] [PubMed] [Google Scholar]
  30. Landschulz, W.H. , Johnson, P.F. and McKnight, S.L. (1988) The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science, 240, 1759. [DOI] [PubMed] [Google Scholar]
  31. Lee, S. , Kim, J. , Han, J.‐J. , Han, M.‐J. and An, G. (2004) Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS‐LIKE 20 (SOC1/AGL20) ortholog in rice. Plant J. 38, 754–764. [DOI] [PubMed] [Google Scholar]
  32. Lee, Y.‐S. , Jeong, D.‐H. , Lee, D.‐Y. , Yi, J. , Ryu, C.‐H. , Kim, S.L. , Jeong, H.J. et al (2010) OsCOL4 is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB . Plant J. 63, 18–30. [DOI] [PubMed] [Google Scholar]
  33. Lee, Y.‐S. , Lee, D.‐Y. , Cho, L.‐H. and An, G. (2014) Rice miR172 induces flowering by suppressing OsIDS1 and SNB, two AP2 genes that negatively regulate expression of Ehd1 and florigens. Rice, 7, 31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lin, R. , Ding, L. , Casola, C. , Ripoll, D.R. , Feschotte, C. and Wang, H. (2007) Transposase‐derived transcription factors regulate light signaling in Arabidopsis . Science, 318, 1302–1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liu, H. , Gu, F. , Dong, S. , Liu, W. , Wang, H. , Chen, Z. and Wang, J. (2016) CONSTANS‐like 9 (COL9) delays the flowering time in Oryza sativa by repressing the Ehd1 pathway. Biochem. Biophys. Res. Commun. 479, 173–178. [DOI] [PubMed] [Google Scholar]
  36. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real‐time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402–408. [DOI] [PubMed] [Google Scholar]
  37. Luan, W. , Chen, H. , Fu, Y. , Si, H. , Peng, W. , Song, S. , Liu, W. et al (2009) The effect of the crosstalk between photoperiod and temperature on the heading‐date in rice. PLoS One, 4, e5891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsubara, K. , Yamanouchi, U. , Wang, Z.‐X. , Minobe, Y. , Izawa, T. and Yano, M. .(2008) Ehd2 a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up‐regulating Ehd1 . Plant Physiol. 148, 1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Matsubara, K. , Yamanouchi, U. , Nonoue, Y. , Sugimoto, K. , Wang, Z.‐X. , Minobe, Y. and Yano, M. (2011) Ehd3, encoding a plant homeodomain finger‐containing protein, is a critical promoter of rice flowering. Plant J. 66, 603–612. [DOI] [PubMed] [Google Scholar]
  40. Meng, X.B. , Zhao, W.S. , Lin, R.M. and Wang, M . (2005) Identification of a novel rice bZIP‐type transcription factor gene, OsbZIP1, involved in response to infection of Magnaporthe grisea . Plant Mol. Biol. Reporter, 23, 301–302. [Google Scholar]
  41. Miao, J. , Guo, D. , Zhang, J. , Huang, Q. , Qin, G. , Zhang, X. , Wan, J. et al (2013) Targeted mutagenesis in rice using CRISPR‐Cas system. Cell Res. 23, 1233–1236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Miernyk, J.A. and Thelen, J.J. (2008) Biochemical approaches for discovering protein–protein interactions. Plant J. 53, 597–609. [DOI] [PubMed] [Google Scholar]
  43. Nemoto, Y. , Nonoue, Y. , Yano, M. and Izawa, T. (2016) Hd1, a CONSTANS ortholog in rice, functions as anEhd1 repressor through interaction with monocot‐specific CCT‐domain protein Ghd7. Plant J. 86, 221–233. [DOI] [PubMed] [Google Scholar]
  44. Nijhawan, A. , Jain, M. , Tyagi, A.K. and Khurana, J.P. (2008) Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice. Plant Physiol. 146, 333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Peng, L.‐T. , Shi, Z.‐Y. , Li, L. , Shen, G.‐Z. and Zhang, J.‐L. (2007) Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter. Biochem. Biophys. Res. Commun. 360, 251–256. [DOI] [PubMed] [Google Scholar]
  46. Ryu, C.‐H. , Lee, S. , Cho, L.‐H. , Kim, S.L. , Lee, Y.‐S. , Choi, S.C. , Jeong, H.J. et al (2009) OsMADS50 and OsMADS56 function antagonistically in regulating long day (LD)‐dependent flowering in rice. Plant, Cell Environ. 32, 1412–1427. [DOI] [PubMed] [Google Scholar]
  47. Sheng, P. , Wu, F. , Tan, J. , Zhang, H. , Ma, W. , Chen, L. , Wang, J. et al (2016) A CONSTANS‐like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice. Plant Mol. Biol. 92, 209–222. [DOI] [PubMed] [Google Scholar]
  48. Shrestha, R. , Gómez‐Ariza, J. , Brambilla, V. and Fornara, F. (2014) Molecular control of seasonal flowering in rice, Arabidopsis and temperate cereals. Annals Botany, 114, 1445–1458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Song, Y.H. , Shim, J.S. , Kinmonth‐Schultz, H.A. and Imaizumi, T. (2015) Photoperiodic flowering: time measurement mechanisms in leaves. Annual Rev. Plant Biol. 66, 441–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Song, S. , Wang, G. , Hu, Y. , Liu, H. , Bai, X. , Qin, R. and Xing, Y. (2018) OsMFT1 increases spikelets per panicle and delays heading date in rice by suppressing Ehd1, FZP and SEPALLATA‐like genes. J. Exp. Botany, 69, 4283–4293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sun, C. , Chen, D. , Fang, J. , Wang, P. , Deng, X. and Chu, C. (2014) Understanding the genetic and epigenetic architecture in complex network of rice flowering pathways. Protein Cell, 5, 889–898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Suzuki, M. , Kao, C.Y. , McCarty, D.R. et al (1997) The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity. Plant Cell, 9, 799–807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tamaki, S. , Matsuo, S. , Wong, H.L. , Yokoi, S. and Shimamoto, K. (2007) Hd3a protein is a mobile flowering signal in rice. Science, 316, 1033. [DOI] [PubMed] [Google Scholar]
  54. Tan, J. , Jin, M. , Wang, J. , Wu, F. , Sheng, P. , Cheng, Z. , Wang, J. et al (2016) OsCOL10, a CONSTANS‐Like gene, functions as a flowering time repressor downstream of Ghd7 in rice. Plant Cell Physiol. 57, 798–812. [DOI] [PubMed] [Google Scholar]
  55. Torok, M. and Etkin, L.D. (2001) Two B or not two B? Overview of the rapidly expanding B‐box family of proteins. Differentiation, 67, 63–71. [DOI] [PubMed] [Google Scholar]
  56. Waadt, R. and Kudla, J. (2008) In planta visualization of protein interactions using bimolecular fluorescence complementation (BiFC). Cold Spring Harbor Protocols, 2008, pdb.prot4995. [DOI] [PubMed] [Google Scholar]
  57. Wei, X. , Xu, J. , Guo, H. , Jiang, L. , Chen, S. , Yu, C. , Zhou, Z. et al (2010) DTH8 suppresses flowering in rice, influencing plant height and yield potential simultaneously. Plant Physiol. 153, 1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wigge, P.A. , Kim, M.C. , Jaeger, K.E. , Busch, W. , Schmid, M. , Lohmann, J.U. and Weigel, D. (2005) Integration of spatial and temporal information during floral induction in Arabidopsis . Science 309, 1056. [DOI] [PubMed] [Google Scholar]
  59. Wu, C. , You, C. , Li, C. , Long, T. , Chen, G. , Byrne, M.E. and Zhang, Q. (2008) RID1 encoding a Cys2/His2‐type zinc finger transcription factor, acts as a master switch from vegetative to floral development in rice. Proc. Natl. Acad. Sci. 105, 12915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xue, W. , Xing, Y. , Weng, X. , Zhao, Y. , Tang, W. , Wang, L. , Zhou, H. et al (2008) Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat. Genet. 40, 761. [DOI] [PubMed] [Google Scholar]
  61. Yano, M. , Katayose, Y. , Ashikari, M. , Yamanouchi, U. , Monna, L. , Fuse, T. , Baba, T. et al (2000) Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS . Plant Cell, 12, 2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhang, Y. , Su, J. , Duan, S. , Ao, Y. , Dai, J. , Liu, J. , Wang, P. et al (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast‐related processes. Plant Methods, 7, 30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhang, L. , Cheng, Z. , Qin, R. , Qiu, Y. , Wang, J.‐L. , Cui, X. , Gu, L. et al (2012) Identification and characterization of an epi‐allele of FIE1 reveals a regulatory linkage between two epigenetic marks in rice. Plant Cell, 24, 4407–4421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang, C. , Liu, J. , Zhao, T. , Gomez, A. , Li, C. , Yu, C. , Li, H. et al (2016) A drought‐inducible transcription factor delays reproductive timing in rice. Plant Physiol. 171, 334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhang, Z. , Hu, W. , Shen, G. , Liu, H. , Hu, Y. , Zhou, X. , Liu, T. et al (2017) Alternative functions of Hd1 in repressing or promoting heading are determined by Ghd7 status under long‐day conditions. Scientific Rep. 7, 5388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhou, F. , Lin, Q. , Zhu, L. , Ren, Y. , Zhou, K. , Shabek, N. , Wu, F. et al (2013) D14‐SCF(D3)‐dependent degradation of D53 regulates strigolactone signalling. Nature 504, 406–410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhu, S. , Wang, J. , Cai, M. , Zhang, H. , Wu, F. , Xu, Y. , Li, C. et al (2017) The OsHAPL1‐DTH8‐Hd1 complex functions as the transcription regulator to repress heading date in rice. J. Exp. Botany, 68, 553–568. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Phylogenetic analysis of BBX family proteins in rice.

Figure S2 OsRE1 physically interacts with OsRIP1.

Figure S3 Expression profiles and subcellular localization of OsRIP1.

Figure S4 OsRIP1 cannot bind to the promoter of Ehd1.in Y1H assay.

Figure S5 Sequencing results of mutant sites in transgenic plants.

Figure S6 Rhythmic expression patterns of genes related to heading date in Nipponbare, Osre1 or Osrip1 under LD conditions.

Figure S7 Interaction relationships between OsRE1, OsRIP1 and several known Ehd1 repressors.

Table S1 Phenotypic data for Nipponbare, Osre1 and Osrip1 mutants.

Table S2 Primers used in this study.

Table S3 Detailed information for rice BBX family members.

Click here for additional data file. (1.6MB, pdf)

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