OsCOL10, a CONSTANS-Like Gene, Functions as a Flowering Time Repressor Downstream of Ghd7 in Rice.

Plant and Cell Physiology Advance Access published February 12, 2016

Title: OsCOL10, a CONSTANS-like gene, functions as a flowering-time repressor downstream of Ghd7 in rice

Running Title: Role of OsCOL10 in the photoperiodic flowering

Corresponding author: Jianmin Wan

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese

E-mail: [email protected]

Telephone: +86-10-62186628

Fax: +86-10-82105837

Subject Areas: (1) growth and development

(3) regulation of gene expression Black and white figures: 3 Color figures: 6 Tables: 0 Supplementary material: 2 tables and 13 figures

© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]

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Academy of Agricultural Sciences, Beijing 100081, China

Title: OsCOL10, a CONSTANS-like gene, functions as a flowering-time repressor downstream of Ghd7 in rice

Running Head: Role of OsCOL10 in the photoperiodic flowering

Junjie Tana,

c, 1

, Mingna Jina, Jiachang Wangb, Fuqing Wua, Peike Shenga, c, Zhijun Chenga, Jiulin Wanga,

Xiaoming Zhenga, Liping Chenb, Min Wanga, Shanshan Zhua, Xiuping Guoa, Xin Zhanga, Xuanming Liuc,

a

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese

Academy of Agricultural Sciences, Beijing 100081, P.R. China

b

National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University,

Nanjing 210095, P.R. China

c

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University,

Changsha 410082, China 1

Present address: Max Planck Institute for Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany

*To whom correspondence should be addressed.

Abbreviations

CO: CONSTANS; FT: FLOWERING LOCUS T; Hd3a: Heading date 3a; RFT1: RICE FLOWERING LOCUS T1; Hd1: Heading date 1; Ehd1: Early heading date 1; Ghd7: Grain Number, Plant Height, and Heading Date7; LD: long day; SD: short day; CRISPR: Clustered, regularly interspaced, short palindromic repeats; NILs: near-isogenic lines;


Chunming Wangb, Haiyang Wanga, Chuanyin Wua, Jianmin Wana, b*

Abstract

Flowering time, or heading date, is a critical agronomic trait that determines the cropping season and regional adaptability, and ultimately grain yield in rice. A number of genes involved in photoperiodic flowering of have been cloned and their roles in modulating expression of the flowering genes have been characterized to a certain extent. However, much less is known about the pathway in transmitting the day length response signal(s) to induce transition to reproductive growth. Here, we report a constitutive flowering repressor OsCOL10, which

(driven by a strong promoter or by fusing it to the activation domain of VP64) showed delayed flowering time under both short and long days. OsCOL10 is affected by the circadian clock and preferentially expressed in leaf mesophyll cells; it is localized to the nucleus and has transcriptional activation activity. Further studies show that OsCOL10 represses the expression of the FT-like genes RFT1 and Hd3a through Ehd1. Transcripts of OsCOL10 are more abundant in the plants carrying a functional Ghd7 allele or over-expressing Ghd7 than in the Ghd7-deficient ones, thus placing OsCOL10 downstream of Ghd7. Together, we conclude that OsCOL10 functions as a flowering-time repressor that links between Ghd7 and Ehd1 in rice.

Key words: Ehd1, Flowering regulation, Heading date, Oryza sativa, photoperiod response


encodes a member of the CONSTANS-like (COL) family. Transgenic rice plants overexpressing OsCOL10

Introduction

Flowering is a complex transition from the vegetative phase to reproductive phase in plants, and is triggered by both endogenous and environmental signals, such as light and temperature (Song et al. 2013). Among environmental signals, the photoperiod or day length is one of the most important regulators of flowering and is perceived by plant leaves (Thomas 1997; Hayama et al. 2003; Song et al. 2013). Based on their response to photoperiod, plants generally fall into three classes: long-day (LD) and short day (SD) plants, whose flowering

Flowering time (also called heading date) in crops is a key agronomic trait that determines the cropping season and regional adaptability. Control of flowering time has been extensively studied for more than 100 years (Jung and Muller 2009).

Numerous genetic and molecular analyses of photoperiodically controlled flowering have been undertaken in the model long-day plant Arabidopsis have established the GIGANTEA(GI)-CONSTANS(CO)-FLOWERING LOCUS T(FT) pathway, where GI integrates cellular signals from light sensory transduction and the circadian clock, and then activates CO, a plant-specific CCT (CO, CO-like, and TOC1) domain transcription activator ( Putterill et al. 1995; Fowler et al. 1999; Park et al. 1999; Yanovsky and Kay 2002). CO promotes flowering through direct activation of FT, which encodes a small mobile protein synthesized in the phloem of leaves. This protein is then transported to the shoot apical meristem (SAM) where it induces expression of floral meristem identity genes (such as AP1 and LEAFY) to initiate transition to reproductive growth (Weigel et al. 1992; Putterill et al. 1995; Kardailsky et al. 1999; Kobayashi et al. 1999; Samach et al. 2000; Abe et al. 2005; Wigge 2006; Corbesier et al. 2007; Turck et al. 2008) .

The GI-CO-FT regulatory pathway is conserved in rice, a SD plant (Turck et al. 2008). Under SD, OsGI regulates expression of Heading date 1 (Hd1), the ortholog of CO (Yano et al. 2000). Hd1 positively regulates


times are promoted under LD and SD, respectively, and day-neutral plants, which are insensitive to day length.

transcription of Heading date 3a (Hd3a), an ortholog of FT, and promotes flowering under SD (Kojima et al. 2002; Hayama et al. 2003). Expression of Hd3a is also induced by another flowering activator known as Ehd1 (Early heading date 1), a B-type response regulator that functions independently of Hd1 under SD conditions (Doi et al. 2004). Under LD, Hd1 converts into a flowering suppressor by reducing expression of Hd3a (Hayama et al. 2003). LD suppression of flowering in rice is also mediated by the strong repressor Ghd7 (Grain number, plant height and heading date 7), which encodes a small protein with a CCT motif (Xue et al. 2008). Ghd7 is

In addition, expression of Ghd7 is activated by morning red light under LD and at midnight under SD. Loss-of-function of SE5 (Photoperiod sensitivity 5), encoding a hemeoxygenase involved in phytochrome chromophore biosynthesis, severely abolishes the expression of Ghd7 (Itoh et al. 2010). Ehd1 and Ghd7 have no orthologs in the Arabidopsis genome, suggesting that Ghd7-Ehd1-Hd3a evolved as a rice-specific flowering suppression pathway (Tsuji et al. 2011). However, the mechanism of Ghd7 suppression remains elusive.

Rice is a facultative SD plant and can eventually flower under non-inductive LD conditions. Recently, molecular genetic studies revealed that a ‘LD activation pathway’ exists to regulate flowering, where a key floral activator, RFT1 (RICE FLOWERING LOCUS T1), the closest paralog of Hd3a, plays a central role (Chardon and Damerval 2005; Komiya et al. 2008). It was shown that RFT1 acts as a major LD activator, in contrast to Hd3a, which functions as a major SD activator (Komiya et al. 2008). The expression of RFT1 is induced by Ehd1 and OsMAS50, a homologue of SOC1 (SUPPRESSOR OF OVEREXPRESION CONSTANS 1) (Lee et al. 2004; Komiya et al. 2008; Komiya et al. 2009) . Thus, the OsMADS50-Ehd1-RFT1 pathway is involved in floral activation under LD conditions.

Accumulating data indicate that Ehd1 is a critical integrator of multiple flowering signals and is regulated by various factors, including multiple positive regulators, such as OsMADS51 under SD, OsMADS50 and DTH2


up-regulated under LD and represses expression of Ehd1 and thus the downstream gene Hd3a (Xue et al. 2008).

(Days to heading 2) under LD, and Ehd2, Ehd3, Ehd4 (Early heading date2, 3, 4) under both SD and LD, and negative regulators, such as Ghd7, DTH8, DTH7/OsPRR37/Ghd7.1 and OsCO3 under either SD or LD, and OsphyB and OsCOL4 in a day length-independent manner ( Izawa et al. 2002; Lee et al. 2004; Kim et al. 2007,2008; Matsubara et al. 2008; Xue, et al. 2008; Komiya et al. 2009; Lee et al. 2010; Wei et al. 2010; Matsubara et al. 2011; Gao et al. 2013; Koo et al. 2013; Wu et al. 2013; Yan et al. 2013; Gao et al. 2014).

Previous studies have showed that the CONSTANS-like (COL) gene family plays critical roles in regulating

flowering repressor under SD and LD, and additionally as a positive regulator of red light signaling and root growth (Datta et al. 2006). AtCOL9 attenuates flowering under LD and AtCOL5 works as a flowering activator under SD (Cheng and Wang 2005; Hassidim et al. 2009). HvCO9 regulates photoperiodic flowering in barley under SD (Kikuchi et al. 2012). The rice genome contains more than 16 COL members ( Robson et al. 2001; Griffiths et al. 2003). Although some of them were shown to be regulators of the photoperiodic flowering, including Hd1, OsCO3, OsCOL4, Ghd7 and DTH2 (Yano et al. 2000; Kim et al. 2008; Xue et al. 2008; Lee et al. 2010; Wu et al. 2013), the remaining ones are not characterized. In this study, we show that transgenic rice plants overexpressing OsCOL10 or the OsCOL10-VP64 fusion display delayed flowering under both SD and LD. We also show that OsCOL10 down-regulates expression of the florigen genes Hd3a and RFT1 through the flowering integrator Ehd1. Moreover, we demonstrated that expression of OsCOL10 is positively regulated by Ghd7, a key LD-specific flowering suppressor. Our findings identify OsCOL10 as a constitutive and Ghd7-regulated flowering repressor in rice.


flowering time in Arabidopsis and other diverse plant species. For example, Arabidopsis COL3 acts as a

Results

OsCOL10-VP64 overexpression causes a delayed flowering phenotype under SD and LD

VP64 is a strong transcriptional activator that has been used to study functionally redundant plant transcription factors (Beerli et al. 1998; Parcy et al. 2002; Silveira et al. 2007). To gain insight into the regulation mechanism underlying photoperiodic flowering in rice, we carried out a large-scale gain-of-function screening for flowering

(TFs-VP64) under control of the maize Ubiquitin (UBI) promoter. This allowed us to overexpress the resulting constructs in a photoperiod-insensitive japonica rice cultivar (cv.) Kita-ake (Figure 1A). Such fusions (TFs-VP64) in this system were previously shown to cause strong dominant effects and, moreover, were useful in studies of redundantly acting plant transcription factors (Beerli et al. 1998; Parcy et al. 2002; Zhang 2003; Silveira et al. 2007; Ohmori et al. 2009; Hanano and Goto 2011). Through phenotypic characteristics and genomic PCR analysis, we isolated more than 50 TFs that changed flowering time upon activation. Among them, a CONSTNAS-like protein, OsJ (Os03g50310) (Robson et al. 2001; Griffiths et al. 2003), delayed flowering time significantly after fusion with VP64. We renamed OsJ as OsCOL10 and further studied its role in controlling photoperiodic flowering.

We selected two independent homozygous transgenic lines (#4 and #7) showing increased accumulation in both mRNA and protein levels of OsCOL10 from a T2 population (Figure 1B, C). We next investigated growth of the two lines under a natural long day (NLD) conditions. During the vegetative stage, growth of the transgenic plants was visually indistinguishable from wild-type plants (Figure 1D). When transition from the vegetative to productive phase took place with emergence of young panicles in wild-type, the transgenic plants remained vegetative and continued to generate new leaves (Figure 1E). The longer vegetative growth phase allowed the transgenic lines to grow taller, produce larger panicles and more seeds than wild-type plants (Figure 1F and


time-associated transcriptional factors (TFs) by fusing various rice TFs to the VP64 activation domain

Figure S1). We quantitatively compared several agronomic traits, including tiller number, plant height, panicle length, grain number per panicle, 1000-grain weight, yield per plant, and plot yield, between wild-type and transgenic plants under NLD conditions (Table S1). Among these traits, plant height, panicle length, grain number per panicle and grain yield (per plant and plot) in both transgenic lines were significantly higher than in wild-type plants.

Under NLD conditions, the transgenic lines flowered about four weeks (#4, 81.6±1.8 days; #7, 85.5±1.2

responses to day length, we grew the transgenic lines and wild-type under SD (10 h light/14 h darkness) and LD (14 h light/10 h darkness) conditions. The transgenic plants flowered about three weeks later than wild-type plants under both light regimes (Figure 1G). To determine whether the delayed flowering phenotype of transgenic plants was associated with reductions in growth rate, we compared the leaf emergence rates of the transgenic lines and wild-type. As shown in Figure 1H, wild-type plants flowered after emergence of the ninth leaf under SD and thirteenth leaf under LD. The transgenic plants had a similar leaf emergence rate until the WT plants flowered, indicating that the late flowering phenotype of transgenic plants was due to delayed floral induction rather than retarded in growth rate. Thus, the enhanced production of OsCOL10-VP64 caused delayed flowering under NLD, SD and LD. We conclude that OsCOL10-VP64 is a constitutive flowering suppressor.

To further confirm the role of OsCOL10 in photoperiodic control of flowering time, we created another overexpression construct (Pubi::OsCOL10) by placing the full-length coding sequence under control of the maize UBI promoter (Figure 2A). Three independent T2 lines (OE4, OE9 and OE14) with increased OsCOL10 expression showed delayed flowering phenotypes under NLD conditions (Figure 2B-D). Under SD conditions (10 h light/ 14 h darkness), we observed that as the wild-type plants were maturing, the OsCOL10-overexpressing plants were still at the stem elongation stage (Figure 2E). These results further support


days) later than the wild-type plants (56.1±1.5 days). To test whether the transgenic lines have different

the notion that OsCOL10 contributes to flowering repression.

To investigate effect of down-regulation of OsCOL10 on flowering time, RNA interference (RNAi)-mediated OsCOL10 knockdown plants were produced. Three independent transgenic lines (RNAi-1, RNAi-2 and RNAi-3) with decreased OsCOL10 expression showed similar flowering times to the wild-type (Figure S2). To exclude the possibility that reduced expression in RNAi lines is also sufficient for plant growth, we generated oscol10 mutants by the CRISPR-Cas method (Miao et al. 2013) and obtained homozygous oscol10

position 410 bp from the ATG start codon, resulting in a frame shift (Figure S3A). We then compared flowering times among wild-type and the mutant lines and there were no significant differences in flowering time between wild-type and mutant lines (Figure S3B and C), suggesting that OsCOL10 shared functional redundancy with other gene(s) in floral regulation.

OsCOl10 is a circadian clock-regulated gene and mainly expressed in the leaves

CO is a member of the CONSTANS-like (COL) gene family, which is characterized by a CCT domain near the carboxy terminus (Putterill et al. 1995; Griffiths et al. 2003). In Arabidopsis, COL proteins can be classified into three groups based on the structure of the B-box near the amino terminus. Group I members (CO and AtCOL1AtCOL5) possess two B-boxes; Group II (AtCOL9-AtCOL15), one B-box and a zinc-finger domain; and Group III (AtCOL6-AtCOL8 and AtCOL16), one B-box (Griffiths et al. 2003). In rice, there are at least 16 members of the COL family that similarly fall into the three main phylogenetic clades (Griffiths et al. 2003; Huang et al. 2012; Figure S4). OsCOL10 belongs to group II, and none of them has been functionally characterized (Figure S4). It is predicted that OsCOL10 contains a B-box domain at the N-terminus and a CCT domain at the C-terminus. A BLAST search with the OsCOL10 protein sequence revealed close homologs in Setaria italica, Sorghum bicolor, Zea mays, Hordeum vulgare and Brachypodium distachyon (Figure S5). Unfortunately, the


mutant lines, oscol10-1, oscol10-2 and oscol10-3. In the oscol10-1 mutant, a 1 bp insertion was present at

functions of these homologs were largely unknown. We examined the temporal expression pattern of OsCOL10 using the samples collected from rice plants grown under SD and LD conditions. The expression of OsCOL10 showed a circadian oscillation (Figure 3A). Under LD, the abundance of OsCOL10 mRNA was low at night, and increased at dawn with a peak in expression after 4 h. Under SD, the abundance of OsCOL10 was low at night and increased at 4 h before dawn with a peak of expression at the dawn (Figure 3A). The rhythmic amplitude of COL10 expression was higher in

growth from the fourth week after germination and reached a peak at the sixth week under SD, whereas the accumulation under LD was delayed to the sixth week and peaked at the eighth week (Figure 3B).

To further determine whether OsCOL10 expression is regulated by the circadian clock, wild-type plants were first grown in cycles of 12 h light/12 h darkness for 30 days and then transferred to continuous light (LL) or continuous darkness (DD). Remarkably, the rhythmic amplitude of OsCOL10 expression was drastically reduced, but continued oscillating for a time after the plants were moved into DD or LL (Figure 3C, D). The results indicate that expression of OsCOL10 is under circadian clock control.

According to the data submitted to the microarray transcript profiling database RiceXPro (http://ricexpro.dna.affrc.go.jp/, Locus ID: Os03g0711100), OsCOL10 was mainly expressed in leaves. To verify

these data, we analyzed the spatial expression pattern of OsCOL10 by qRT-PCR using RNA samples from various tissues and leaves at different developmental stages (Figure 4A). OsCOL10 was expressed preferentially in the leaves, especially in young developing leaves (Figure 4B). Histochemical staining of transgenic plants carrying the GUS reporter gene driven by the OsCOL10 promoter supported the above expression pattern and further delimited GUS activity to cells outside the vascular tissues (Figures 4C-J). In situ hybridization assays confirmed that OsCOL10 was enriched in leaf mesophyll cells (Figure 4K, L).


plants grown under SD than under LD (Figure 3A). Moreover, OsCOL10 started to accumulate during vegetative

OsCOl10 may act as a transcriptional regulator

The CCT domain of OsCOL10 suggests that it is likely localized in the nucleus (Robson et al. 2001; Zhang et al. 2015). To confirm that OsCOL10 is a nuclear protein, a translational fusion of OsCOL10 and green fluorescence protein (GFP) under control of the CaMV 35S promoter (35S:OsCOL10-GFP) was constructed. A transient expression assay in onion epidermis cells was used to assess the subcellular localization of the fusion protein. In cells bombarded with expression of the fusion protein, signal of GFP fluorescence was localized in the nucleus;

(Figure 5A-F). Nuclear localization of OsCOL10 was further confirmed by observation of the OsCOL10-GFP fusion protein in stable transgenic rice root cells (Figure 5G-I). These results confirm that OsCOL10 is localized in the nucleus.

We next investigated whether OsCOL10 has transcriptional activation activity. As shown in Figure 5J, full-length OsCOL10 had weak transcriptional activation activity in yeast cells. Deletion analysis showed that neither the B-box zinc finger domain nor the CCT domain alone could induce transcriptional activation activity; rather, the middle region between them was essential in a similar way to CO and other previously reported CO-like proteins (Tiwari et al. 2010; Wu et al. 2013). These results support the notion that OsCOL10 may have an important role in transcriptional regulation of downstream gene expression.

OsCOL10 delayed flowering time mainly through repression of Ehd1

In rice, Hd3a and RFT1, known as florigen genes, are required for floral induction (Kojima et al. 2002; Tamaki et al. 2007; Komiya et al. 2008). To understand the molecular mechanism by which OsCOL10 contributes to flowering repression, we determined the transcriptional levels of Hd3a and RFT1 in wild-type and transgenic plants by qRT-PCR. The expression levels of Hd3a and RFT1 were diminished in transgenic plants


whereas in cells expressing free GFP, fluorescence of GFP could be detected in both the nucleus and cytoplasm

overexpressing modified OsCOL10 (OsCOL10-VP64) and OsCOL10 under both SD and LD conditions (Figure 6B, C, G and H; Figure S6B, C, G and H). The transcript abundances of downstream floral meristem identity genes, such as OsMADS14 and OsMADS15, were also reduced in the transgenic plants (Figure S7A, B, K and L; Figure S8A, B, K and L).

Transduction of floral signals in rice mainly occurs through two key floral integrators, Hd1 and Ehd1 (Tsuji et al. 2011). To understand whether OsCOL10-regulated expression of the two florigen genes is mediated by Hd1,

and LD conditions. Interestingly, the abundance of Ehd1 was reduced in the transgenic plants, whereas the expression level of Hd1 was similar in transgenic and wild-type plants (Figure 6D, E, I and J; Figure S6D, E, I and J). In transgenic plants, where OsCOL10(itself or fused to VP64) is under control of the maize Ubiquitin (UBI) promoter, the expression of OsCOL10 maintain relatively high levels at each time point which contributes to enhance the repression of Ehd1(Figure 6A, F, D and I; Figure S6A, F, D and I). These results suggest that OsCOL10 regulates Hd3a and RFT1 expression through Ehd1. We also examined the expression of other known flowering regulators in transgenic and wild-type plants and found that transcriptional levels were not affected in the transgenic plants (Figure S7 and Figure S8). These findings suggest that OsCOL10 functions are not upstream of the known floral regulators.

To further verify that OsCOL10 suppresses Hd3a and RFT1 through Ehd1, we used qRT-PCR to compare expression levels of the 3 genes in wild-type and transgenic plants during a 10-week growth period. Transcription levels of Ehd1, Hd3a and RFT1 in transgenic plants were significantly reduced in comparison with those in wild-type plants from 2 weeks after germination (Figure 7), thus further supporting the notion that OsCOL10 down-regulates expression of Ehd1 and then the two downstream florigen genes (Hd3a and RFT1).

To further understand whether OsCOL10 regulated transcription of Ehd1 in a direct manner, a yeast


Ehd1, or both, we compared the mRNA levels of these two genes in wild-type and transgenic plants under SD

one-hybrid assay was performed. The results showed that OsCOL10 could not directly bind to the promoter region of Ehd1 (Figure S9).

Expression of OsCOL10 is regulated by Ghd7

To determine whether OsCOL10 is regulated by known flowering genes we examined the circadian expression of OsCOL10 in near isogenic lines (NILs) carrying defective Hd1, Hd2, Ehd1, Hd3a, DTH2, DTH7, DTH8 or

OsCOL10 was detected with no significant differences from wild-type counterparts in most of the mutants and NILs except for the NILs of Ghd7 (Figure S10 and Figure 8A, B). Additionally, there was no significant change of the OsCOL10 expression observed in NILs deficient in either Hd3a or Ehd1 (Figure S10G, I).

Abundance of OsCOL10 transcripts in the NIL carrying a defective allele of Ghd7 (NIL [ghd7]) was lower than that in the NIL carrying a functional allele of Ghd7 (NIL [Ghd7]) under LD conditions, suggesting that Ghd7 regulates its expression under LD. Given that wild-type Kita-ake plants carry a defective Ghd7 allele (Gao et al. 2013), we introduced the functional full-length Ghd7 coding sequence driven by its native promoter from japonica cv. Nipponbare into Kita-ake to create a Ghd7/ghd7 pair of a different genetic background. Transgenic Ghd7 and non-transgenic plants amplified distinctly different PCR fragments amplified with primers flanking the Ghd7 intron (Figure 8C). The transgenic plants exhibited a late-flowering phenotype relative to the wild-type (Figure 8D, E) under NLDs. We then compared the expression pattern of OsCOL10 between the transgenic and wild-type plants under LD. As expected, the mRNA transcripts of OsCOL10 became more abundant as a result of the introduction of the functional Ghd7 allele (Figure 8F). Since Ghd7 expression was severely impaired in the se5 mutant (Itoh et al. 2010), we also investigated the circadian expression of OsCOL10 in se5 in the background of Nipponbare (Sun et al. 2012). The abundance of OsCOL10 transcript was significantly reduced in se5 compared to that in wild-type (Figure 8G). To determine whether such a reduction in OsCOL10 expression


Ghd7 alleles, as well as in OsphyA, OsphyB, and OsphyC mutants under SD and LD conditions. Expression of

was in a Ghd7-mediated manner, we used another se5 mutant in the background of Gang46B (Chen et al. 2013), where Ghd7 activity is absent (Figure S11). Differences in expression of OsCOL10 wild-type and se5 plants were not significant (Figure 8H), suggesting that the reduced expression of OsCOL10 in this se5 mutant resulted from lack of Ghd7 expression, thus further supporting involvement of Ghd7 in regulation of OsCOL10. Ghd7 was reported to function as a transcription factor (Xue et al. 2008). A yeast one-hybrid assay was performed to determine whether transcription of OsCOL10 was directly regulated by Ghd7. The result indicated that Ghd7

expression of OsCO3 and OsCOL4, other two CO-like family members acting as flowering repressors, as well as OsK and OsL, close to OsCOL10 in the phylogenetic tree of Group II, in the Ghd7 NIL and transgenic plants, together with the corresponding controls. No significant differences were detected (Figure S13). Thus the overall results suggest that Ghd7 regulates OsCOL10 specifically under LD.

Discussion

OsCOL10 is a constitutive flowering repressor under SD and LD conditions In this study we characterized a constitutive flowering repressor, OsCOL10 that was previously identified as a member of the CONSTNAS- like gene family (Griffiths et al. 2003; Huang et al. 2012; Zhang et al. 2015). Although some members involved in photoperiodic control of flowering time had been reported in rice, OsCOL10 acts differently from most of these CO-like members because it represses flowering independently of day length. For example, Hd1, the first identified member and an ortholog of CO, functions as a flowering promoter in SD but a repressor in LD (Yano et al. 2000); OsCO3 negatively regulates flowering under SD conditions (Kim et al. 2008); Ghd7 is a LD-specific flowering repressor (Xue et al. 2008); and Days to heading 2 (DTH2), encoding for a CO-like protein, works as a LD-specific flowering inducer (Wu et al. 2013). OsCOL4 was known as the only CO-like member that repressed flowering under both SD and LD conditions (Lee et al.


could not bind directly to the promoter region of OsCOL10 (Figure S12). We also measured the circadian

2010). Although OsCOL10 and OsCOL4 have many similarities in regulating flowering in rice, such as flowering repression by reducing the expression of Ehd1, they are nevertheless involved in different photoperiodic regulatory pathways. OsCOL4 acts downstream of OsphyB, another constitutive flowering repressor, and thus the OsphyB-OsCOL4 regulatory pathway is shared in SD and LD (Lee et al. 2010). By contrast, OsCOL10 is not regulated by phytochrome genes and has different regulatory mechanisms under SD and LD (Figure S10; Figure 9). Expression of OsCOL10 peaked at dawn in SD and 4 h after dawn in LD

and I), suggesting that OsCOL10 may have a role in switching-off Ehd1 expression in a circadian manner. OsCOL10 RNAi plants and CRISPR-Cas-induced oscol10 mutants showed no visible phenotypic differences suggesting that, like other CO-like gene(s), OsCOL10 shared functional redundancy in floral regulation. Similar results were reported in CO-like studies in Arabidopsis. For example, overexpression of ATCOL5 or ATCOL9 promoted or repressed flowering, respectively, but their mutants showed no significant changes in phenotype (Cheng and Wang 2005; Hassidim et al. 2009). These findings demonstrated that CO-like family members act distinctly and redundantly to coordinate regulation of flowering. The mechanism, by which this occurs, needs to be further elucidated. Plants overexpressing a modified OsCOL10 (OsCOL10-VP64) or OsCOL10 itself displayed parallel delayed flowering under SD and LD conditions, indicating that OsCOL10-VP64 acts like the native OsCOL10, suggesting that the native OsCOL10 functions as a transcriptional activator or a carrier of an activator to promote the transcription of unknown downstream flowering repressors. It is noteworthy that OsCOL10 has only weak transcriptional activation activity in yeast cells (Figure 5J). We propose that OsCOL10 might act as a mediator in regulatory complexes. In other words, OsCOL10 may interact with a co-activator to participate in transcriptional activation. However, we failed to isolate such candidate proteins by yeast two-hybrid screening, and their


whereas Ehd1 expression peaked at 4 h before and after dawn in SD and LD, respectively (Figure 3A; Figure 6D,

identification remains an important task for future studies. In addition, OsCOL10 represses Ehd1 probably in an indirect fashion (Figure S9), suggesting the possibility that OsCOL10 activates an unknown factor which represses Ehd1 (Figure 9). In OsCOL10-VP64 plants, expression levels of Hd3a and RFT1 under SD and LD were almost completely repressed until 10 weeks after germination (Figure 7). However, they flowered around 12 weeks after germination (Figure 1). A possible explanation is that the samples in Figure 7 were taken at dawn while

accumulation at peak point may be capable of inducing flowering.

OsCOL10 is regulated by Ghd7 under LD Flowering in rice is inhibited under LD conditions. One of the major contributors to LD-dependent flowering repression in rice comes from the presence of a small CCT domain protein, Ghd7, which not only acts as a key negative regulator of flowering, but also has crucial roles in promoting productivity and adaptability (Xue et al. 2008). Rice cultivars with impaired function of Ghd7 (Ghd7-0 or Ghd7-0a) with reduced sensitivity to photoperiod flower very early even in LD conditions (Xue et al. 2008). It was shown that Ghd7-associated repression of the flowering time was mainly through the suppression of flowering promoter Ehd1 ( Xue et al. 2008; Komiya et al. 2009). In addition, repression of Ehd1 by Ghd7 is critical for rice to recognize the critical day-length (Itoh et al. 2010). Although some genes were reported to regulate the expression of Ghd7 such as SE5 and Ehd3 (Itoh et al. 2010; Matsubara et al. 2011), the mechanism by which Ghd7 represses the Ehd1 is still unknown. In the current study, we obtained evidence to show that expression of OsCOL10 is regulated by Ghd7 (Figure 8). Both Ghd7 and OsCOL10 act as repressors of Ehd1. Therefore, OsCOL10 establishes a functional link between Ghd7 and Ehd1 in the photoperiodic control of flowering. It is noteworthy that expression of OsCOL10 is still abundant and rhythmic when Ghd7 is deficient (Figure 8A, F), indicating that Ghd7 is not the


expressions of Hd3a and RFT1 peak at 4 h after down under SD and LD (Figure 6), thus their expression

sole regulator of OsCOL10. Thus, we speculated that OsCOL10 is diurnally regulated by another unknown flowering regulator(s) under LD (Figure 9). In addition, repression of Ehd1 by Ghd7 is not only mediated by OsCOL10, because OsCOL10 RNAi and oscol10 plants showed no visible phenotypes (Figure S2 and Figure S3) and the effect of OsCOL10 on flowering in rice is far less than Ghd7. These findings lead us to believe that there are at least two pathways through which Ghd7 represses the expression of Ehd1 (Figure 9). In other words, the final repression effect of Ehd1 through Ghd7 comes from the combined effects of these pathways. Identification

Ghd7 is induced by phytochromes, and its expression is severely reduced in se5 mutants (Itoh et al. 2010). In addition, SE5 repressed the expression of Ehd1 under both SD and LD (Andres et al. 2009). SE5 suppression of Ehd1, can be mediated by Ghd7 (Itoh et al. 2010), but we also showed that SE5 represses Ehd1 independently of Ghd7 in LD (Figure S11D), because the expression of Ehd1 is still significantly increased in the se5 mutant with the background of G46B where Ghd7 is not functional. Therefore, there are two pathways through which SE5 suppresses the expression of Ehd1 under LD: a Ghd7- dependent pathway and a Ghd7-independent pathway (Figure 9). Our results showed that OsCOL10 can be regulated by SE5 in a Ghd7-mediated manner under LDs (Figure 8G and H). Therefore, OsCOL10 is involved in the Ghd7-dependent pathway, whereas the previously indentified CO-like gene, OsCOL4, which functions between OsPHYB and Ehd1, is implicated in the other pathway.

OsCOL10 was preferentially expressed in leaf mesophyll cells (Figure 4K, L), whereas Ghd7 mainly accumulated in vascular tissues (Xue et al. 2008). Moreover, yeast one-hybrid assays showed that Ghd7 does not directly regulate the expression of OsCOL10 (Figure S12), implying that OsCOL10 is not the direct target of Ghd7, and that other unidentified proteins must transmit the signal to mediate the regulatory machinery.

Materials and Methods


of the other pathway(s) is still a challenge for future studies.

Plant material and growth conditions

Overexpression lines for OsCOL10 fused or not fused to VP64 were generated in Oryza sativa japonica cv. Kita-ake. All plants were grown in a paddy field in Beijing (40°13’N, 116°13’E, summer the summer) or in controlled chambers under SD (10 h light at 30℃/14 h darkness at 25℃) or LD(14 h light at 30℃/10 h dark at 25℃) with a relative humidity of ～70% and light intensity of ～800 µmol m-2s-1.

non-functional DTH7 and a NIL carrying a functional DTH7 allele; Asominori carrying a functional DTH2 allele and a NIL carrying a non-functional DTH2 allele; Nipponbare carrying a functional Hd1 allele and a NIL carrying a non-functional Hd1 allele; Nipponbare carrying a functional Hd2 allele and a NIL carrying a non-functional Hd2 allele; Nipponbare carrying a partially functional Hd3a allele and a NIL carrying a functional Hd3a allele; Taichun 65 carrying a non- functional Ehd1 allele and a NIL carrying a functional Ehd1 allele; Asominori carrying a functional DTH8 allele; and a NIL carrying a non-functional DTH8 allele; and a NIL pair carrying functional or non-functional Ghd7 allele in Zhenshan97 background. Vector construction and transformation

To create the overexpression construct Pubi:: OsCOL10-VP64, the OsCOL10 full-length cDNA driven by the maize Ubiquitin (Ubi) gene was cloned into the binary vector CVP64B using the Gateway cloning system. For Pubi::OsCOL10, OsCOL10 full-length cDNA driven by the maize Ubi promoter was cloned into binary vector pCAMBIA1390 using In-Fusion Advantage PCR Cloning Kits (Clontech). To generate a pGhd7::Ghd7 construct, the encoding sequence of Ghd7 driven by its native promoter was cloned into pCAMBIA-1305.1. The resulting plasmids were transformed into Kita-ake plants by A. tumefaciens-mediated transformation as described previously (Hiei et al. 1994).


For gene expression analyses, the following accessions and their NILs were used: Kita-ake carrying a

For RNAi, the construct pCUbi1390-∆FAD2 (Ubiquitin promoter and a FAD2 intron inserted into pCAMBIA1390) was used as an RNAi vector (Tan et al. 2014). Both antisense and sense versions of a specific 153 bp fragment from the coding region were amplified (primer pairs COL10RNAi-1 and COL10RNAi-2, Table S2), and inserted into pCUbi1390-∆FAD2 to form the RNAi construct pUbi-dsRNAi- COL10, which was then transformed into japonica cv. Nipponbare by the above method. The CRISPR-Cas system was used as described previously (Miao et al. 2013) to obtain oscol10 mutant

followed by ligation into a binary vector containing the Cas9 expression cassette using the Gateway cloning system. The resulting combination vector was then transformed into cv. Kita-ake as described above. All primer sequences are listed in Table S2.

RNA preparation and qRT-PCR analysis

Total rice RNA was extracted with an RNA Prep Pure Kit (Zymo Research, Orange, CA) according to the manufacturer’s instructions. Then, 20 µL cDNA was synthesized using 1µg RNA with the QuantiTect Reverse Transcription Kit (QIAGEN). Primer pairs (Table S2) were designed using Primer Express (Applied Biosystems). qRT-PCR was conducted using SYBR Premix Ex Taq Kit (TaKaRa; RR041A) in an ABI PRISM 7900HT (Applied Biosystems) according to the manufacturer’s instructions. The procedure was: initial polymerase activation for 30 s at 95°C followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The rice Ubiquitin gene was used as the internal control. For each sample, qRT-PCR was performed with three technical and three biological replicates. The 2–∆∆CT method was used to analyze relative transcript levels of gene expression (Livak and Schmittgen 2001).

RNA in situ hybridization.


lines. Briefly, an 18 bp OsCOL10-specific spacer sequence was firstly cloned into entry vector pOs-sgRNA

Five-week-old leaf blades were collected and fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde) overnight at 4°C, followed by a series of dehydration steps, and then embedded in paraffin (Paraplast Plus, Sigma), where RNA in situ hybridization was performed as described previously (Bradley et al. 1993). To prepare the probe, we used a pair of primers, COL10-in situ-F (5′-AGAGGACGACGAGGACCT-3′) and COL10-in situ-R (5′-ATGACTCGCTGGGATCGAA-3′), to amplify a 503 bp unique sequence of OsCOL10 from a cDNA clone. The fragment was then inserted into the pGEM-T vector (Promega) for RNA transcription.

following instructions provided by the manufacturer. The slides were observed under bright field with a Leica DM5000B microscope and photographed with a Leica DFC490 camera.

Subcellular localization of OsCOL10-GFP proteins

The coding sequence of OsCOL10 was ampliﬁed and cloned into the N-terminus of green fluorescent protein (GFP) under control of the Cauliflower mosaic virus (CaMV) 35S promoter in the transient expression vector pA7-GFP, generating recombinant pA7-OsCOL10-GFP. The recombinant vector was then transformed into onion epidermal cells via shotgun bombardment (PDS-1000/He; Bio-Rad). The GFP signal was visualized using a confocal laser-scanning microscope (LSM 700; Carl Zeiss).

Transactivation activity assays

Transactivation activity assays were performed using the Match-maker GAL4 Two-Hybrid System 3 (Clontech). Plasmids containing the GAL4 DNA binding domain fused with OsCOL10 deletions were transformed into yeast strain AH109. The substrate chlorophenol red-b-D-galactopyranoside (CPRG; Roche Biochemicals) was used to measure b-galactosidase activity according to the Yeast Protocols Handbook (Clontech).

Yeast one-hybrid assay


Digoxigenin-labelled RNA probes were prepared using a DIG Northern Starter Kit (Cat. No. 2039672, Roche)

The assay was carried out as described (Gao et al. 2013). To generate AD-Ghd7 and AD-OsCOL10, the full-length Ghd7 and OsCOL10 coding sequences were amplified by RT-PCR from cv. Minhui 63 and Kita-ake, respectively, and ligated into the pB42AD vector (Clontech) digested with EcoRI. To generate OsCOL10p::LacZ and Ehd1p::LacZ reporter genes, various fragments of the OsCOL10 and Ehd1 promoters were amplified from Kita-ake genomic DNA and inserted into corresponding sites in the reporter plasmid pLacZi. Plasmids were co-transformed into yeast strain EGY48. Transformants were grown on SD/Trp-/Ura plates for 48 h and then

Funding

This work was supported by National Natural Science Foundation (grant no. 31401466 and 31371601), and National Transformation Science and Technology Program (2013ZX08009-003). Disclosures The authors have no conflicts of interest to declare.

Acknowledgements

We thank Dr. Masahiro Yano (National Institute of Agrobiological Sciences, Japan) for kindly providing NILs (Hd1 and Hd3a), Dr. Atsushi Yoshimura (Kyushu University, Japan) for mutants (OsphyA, B, C), Dr. Qifa Zhang (Huazhong Agricultural University, Wuhan) for the NIL Ghd7/ghd7 NIL pair, and Dr. Chengcai Chu (Chinese Academy of Sciences, Beijing) for the se5 mutant.

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Figure 1. Overexpression of OsCOL10-VP64 showing delayed flowering time. (A) Schematic diagram of Pubi::TFs-VP64 fusion construct. The tetrameric VP16 activation domain (gray boxes) made up the VP64 domain. Pubi, maize Ubiquitin promoter. (B) qRT-PCR analyses of OsCOL10 in wild-type (WT) and two transgenic lines. Samples were harvested at 4 h after dawn from second leaf blades from top of 55-day-old plants under NLD. (C) Western blot analyses of OsCOL10 in WT and transgenic lines with a VP16-specific


role in grain yield and adaptation in rice. Cell Res. 23: 969-971.

antibody. The same filter was stained with Ponceau S to visualize total protein (bottom). (D-F) Phenotypic characteristics of WT and transgenic lines at the young (D), flowering (E) and mature stages (F); the plants from left to right in every panel are wild-type and two independent transgenic lines (#4 and #7). (G) Comparison of flowering times among WT and two transgenic lines under different day-length conditions. NLD, natural long-day; SD, short-day; LD, long-day. Days to flowering was scored when first panicle emerged. Error bars indicate standard deviations; n = 10-20 plants. (H) Leaf emergence rate of WT and

lines.

Figure 2. Phenotypic characterization of OsCOL10-overexpressing plants. (A) schematic diagram of the Pubi::OsCOL10 construct. Tnos, terminator of nopaline synthase gene (nos). (B) qRT-PCR analyses of OsCOL10 in wild-type (WT) and three independent Pubi::OsCOL10 transgenic lines (OE#4, OE#9 and OE#14). Samples were harvested 4 h after dawn from second leaf blades from the tops of 55-day-old plants grown under NLD. (C) Phenotypes of Pubi::OsCOL10 plants at flowering stages. (D) Comparison of flowering times among WT and transgenic lines under natural long-day conditions. (E) Phenotypic characteristics of WT and transgenic lines under SD when the WT plants showed complete emergence of panicles from the apices. The white boxes indicate the sources of the corresponding magnified images. The first bar is 2 mm and the others are 1 µm.

Figure 3. OsCOL10 is a circadian clock-regulated gene. (A-B) Rhythmic and developmental expression of OsCOL10. In (A), penultimate leaf blades were harvested at indicated time points from 30-day-old (SDs) and 35-day-old (LDs) plants. In (B), penultimate leaf blades were harvested at dawn of the indicated day. Arrowheads represent the flowering time of plants at corresponding growth stages. (C-D) qRT-PCR analysis of diurnal expression patterns of OsCOL10 under LL (C) and DD (D) conditions. The plants were first grown in 12


transgenic plants under both SDs and LDs (n = 20). Arrow indicates the flowering times of corresponding

h light/12 h darkness for 30 days and then transferred to continuous light (LL) or continuous darkness (DD) at dusk. In panels (A), (C) and (D), white bars indicate light; black bars indicate darkness. The gray bars represent the subjective darkness in (C) and subjective light in (D), respectively. Values represent means ± sd from three independent biological replicates. The rice Ubiquitin (UBQ) gene was used as the internal control. sd, standard deviation.

were used for qRT-PCR. DL1, newly emerging leaf; DL2, expanding leaf; DL3, fully expanded leaf; ASA, around the shoot apex. (B) OsCOL10 transcript levels in various organs (means ± sd, n = 3). (C-J) Histochemical staining of various organs in pOsCOL10::GUS transgenic plants. (C), root; (D) stem; (E) Transverse sections of the stem; (F) Magnified image of boxed area in (E); (G and H) Transverse sections of the leaf blade and sheath; (I) enlarged image of boxed area in (H); (J) Floret; (K-L) RNA in situ hybridization analysis of OsCOL10. The materials were harvested at 4 h after dawn under LD conditions. Leaf blades of 35-day-old wild-type plants were cross-sectioned and hybridized with OsCOL10-specific antisense (K) or sense (L) probes. x, xylem; p, phloem; ep, epidermis; m, mesophyll.

Figure 5. OsCOL10 acts as a transcriptional regulator. Subcellular localization of fused OsCOL10-GFP in onion epidermal cells (A-F) and transgenic rice roots cells (G-I). Bars = 50 µm. (J) Transactivation activity assays of OsCOL10 and its deletion derivatives in the yeast GAL4 system. BD, GAL4-DNA binding domain; AD, GAL4 activation domain; ZF, MR, and CCT indicate the zinc finger region, the middle region, and the CCT domain of OsCOL10, respectively; EV denotes empty vector. β-galactosidase (β-gal) activity was measured using a liquid culture assay. Mean values ± SD were collected from three independent experiments.

Figure 6. Rhythmic expression pattern of OsCOL10, Hd3a, RFT1, Ehd1 and Hd1 in the wild-type and


Figure 4. OsCOL10 was preferentially expressed in the leaves. (A) 30-day-old wild-type plants grown under SD

OsCOL10-VP64 transgenic plants under SDs and LDs. SDs (A-E); LDs (F-J). Penultimate leaf blades were harvested at indicated time point from 30-day-old (SDs) and 35-day-old (LDs) wild-type and transgenic plants. The open and filled bars at the bottom represent light and dark periods, respectively. Expression levels are relative to the rice UBQ gene. Values are shown as means ± sd of three independent biological replicates. sd, standard deviation.

and OsCOL10-VP64 plants. Samples were collected from penultimate leaf blades at dawn of the indicated day under SD and LD conditions. Expression levels are relative to that of the rice UBQ gene. Values are shown as means ± sd of three biological replicates. sd, standard deviation.

Figure 8. Expression of OsCOL10 is regulated by Ghd7. (A-B) Comparison of circadian expression of OsCOL10 between the near isogenic lines carrying functional (Ghd7) non-functional (ghd7) alleles under LD (A) and SD (B) conditions. (C) The functional Ghd7 allele was introduced into the Ghd7-deficient rice cv. Kita-ake and transgenic positive plants were checked by PCR with the primers flanking the intron. Red rows represent the two amplified fragments in the transgenic positive plants. (D) Phenotypic characterization of transgenic positive plants under NLD conditions. (E) Comparison of flowering time between the Ghd7 transgenic and wild-type plants under NLD conditions. (F) Circadian expression of OsCOL10 in the wild-type plants and Ghd7 transgenic lines. (G-H) Diurnal expression of OsCOL10 in the wild-type and se5 mutants with the background of Nipponbare (Nip) (G) or Gang46B (G46B) (H) under LD conditions. Penultimate leaf blades were harvested at indicated time point from 30-day-old (SDs) (B) and 35-day-old (LDs) (A, F, and G-H) transgenic plants or mutants and corresponding wild-type plants. Open and filled bars at the bottom in (A-B) and (F-H) represent the light and dark periods, respectively. Expression levels are relative to the rice UBQ gene. Values are means ± sd


Figure 7. Changes in levels of OsCOL10, Ehd1, Hd3a and RFT1 transcripts during development in wild-type

of three independent biological replicates. sd, standard deviations.

Figure 9. A proposed model of OsCOL10 in regulation of photoperiodic flowering time in rice. Blue lines represent pathways in LD and red lines indicate pathways shared between SD and LD. x, y and z represent existing but unknown gene(s). Dashed lines indicate unknown regulatory genes that may function by activation or repression.



Figure 1. Overexpression of OsCOL10-VP64 showing delayed flowering time. (A) Schematic diagram of Pubi::TFs-VP64 fusion construct. The tetrameric VP16 activation domain (gray boxes) made up the VP64 domain. Pubi, maize Ubiquitin promoter. (B) qRT-PCR analyses of OsCOL10 in wild-type (WT) and two transgenic lines. Samples were harvested at 4 h after dawn from second leaf blades from top of 55-day-old plants under NLD. (C) Western blot analyses of OsCOL10 in WT and transgenic lines with a VP16-specific antibody. The same filter was stained with Ponceau S to visualize total protein (bottom). (D-F) Phenotypic characteristics of WT and transgenic lines at the young (D), flowering (E) and mature stages (F); the plants from left to right in every panel are wild-type and two independent transgenic lines (#4 and #7). (G) Comparison of flowering times among WT and two transgenic lines under different day-length conditions. NLD, natural long-day; SD, short-day; LD, long-day. Days to flowering was scored when first panicle emerged. Error bars indicate standard deviations; n = 10-20 plants. (H) Leaf emergence rate of WT and transgenic plants under both SDs and LDs (n = 20). Arrow indicates the flowering times of corresponding lines.

220x288mm (600 x 600 DPI)



Figure 2. Phenotypic characterization of OsCOL10-overexpressing plants. ¬¬¬(A) schematic diagram of the Pubi::OsCOL10 construct. Tnos, terminator of nopaline synthase gene (nos). (B) qRT-PCR analyses of OsCOL10 in wild-type (WT) and three independent Pubi::OsCOL10 transgenic lines (OE#4, OE#9 and OE#14). Samples were harvested 4 h after dawn from second leaf blades from the tops of 55-day-old plants grown under NLD. (C) Phenotypes of Pubi::OsCOL10 plants at flowering stages. (D) Comparison of flowering times among WT and transgenic lines under natural long-day conditions. (E) Phenotypic characteristics of WT and transgenic lines under SD when the WT plants showed complete emergence of panicles from the apices. The white boxes indicate the sources of the corresponding magnified images. The first bar is 2 mm and the others are 1 µm. 189x213mm (300 x 300 DPI)


Figure 3. OsCOL10 is a circadian clock-regulated gene. (A-B) Rhythmic and developmental expression of OsCOL10. In (A), penultimate leaf blades were harvested at indicated time points from 30-day-old (SDs) and 35-day-old (LDs) plants. In (B), penultimate leaf blades were harvested at dawn of the indicated day. Arrowheads represent the flowering time of plants at corresponding growth stages. (C-D) qRT-PCR analysis of diurnal expression patterns of OsCOL10 under LL (C) and DD (D) conditions. The plants were first grown in 12 h light/12 h darkness for 30 days and then transferred to continuous light (LL) or continuous darkness (DD) at dusk. In panels (A), (C) and (D), white bars indicate light; black bars indicate darkness. The gray bars represent the subjective darkness in (C) and subjective light in (D), respectively. Values represent means ± sd from three independent biological replicates. The rice Ubiquitin (UBQ) gene was used as the internal control. sd, standard deviation. 99x59mm (600 x 600 DPI)


Figure 4. OsCOL10 was preferentially expressed in the leaves. (A) 30-day-old wild-type plants grown under SD were used for qRT-PCR. DL1, newly emerging leaf; DL2, expanding leaf; DL3, fully expanded leaf; ASA, around the shoot apex. (B) OsCOL10 transcript levels in various organs (means ± sd, n = 3). (C-J) Histochemical staining of various organs in pOsCOL10::GUS transgenic plants. (C), root; (D) stem; (E) Transverse sections of the stem; (F) Magnified image of boxed area in (E); (G and H) Transverse sections of the leaf blade and sheath; (I) enlarged image of boxed area in (H); (J) Floret; (K-L) RNA in situ hybridization analysis of OsCOL10. The materials were harvested at 4 h after dawn under LD conditions. Leaf blades of 35-day-old wild-type plants were cross-sectioned and hybridized with OsCOL10-specific antisense (K) or sense (L) probes. x, xylem; p, phloem; ep, epidermis; m, mesophyll. 167x268mm (300 x 300 DPI)


Figure 5. OsCOL10 acts as a transcriptional regulator. Subcellular localization of fused OsCOL10-GFP in onion epidermal cells (A-F) and transgenic rice roots cells (G-I). Bars = 50 µm. (J) Transactivation activity assays of OsCOL10 and its deletion derivatives in the yeast GAL4 system. BD, GAL4-DNA binding domain; ZF, MR, and CCT indicate the zinc finger region, the middle region, and the CCT domain of OsCOL10, respectively; EV denotes empty vector. β-galactosidase (β-gal) activity was measured using a liquid culture assay. Mean values ± SD were collected from three independent experiments. 86x94mm (300 x 300 DPI)


Figure 6. Rhythmic expression pattern of OsCOL10, Hd3a, RFT1, Ehd1 and Hd1 in the wild-type and OsCOL10-VP64 transgenic plants under SDs and LDs. SDs (A-E); LDs (F-J). Penultimate leaf blades were harvested at indicated time point from 30-day-old (SDs) and 35-day-old (LDs) wild-type and transgenic plants. The open and filled bars at the bottom represent light and dark periods, respectively. Expression levels are relative to the rice UBQ gene. Values are shown as means ± sd of three independent biological replicates. sd, standard deviation. 167x60mm (300 x 300 DPI)


Figure 7. Changes in levels of OsCOL10, Ehd1, Hd3a and RFT1 transcripts during development in wild-type and OsCOL10-VP64 plants. Samples were collected from penultimate leaf blades at dawn of the indicated day under SD and LD conditions. Expression levels are relative to that of the rice UBQ gene. Values are shown as means ± sd of three biological replicates. sd, standard deviation. 107x153mm (600 x 600 DPI)


Figure 8. Expression of OsCOL10 is regulated by Ghd7. (A-B) Comparison of circadian expression of OsCOL10 between the near isogenic lines carrying functional (Ghd7) non-functional (ghd7) alleles under LD (A) and SD (B) conditions. (C) The functional Ghd7 allele was introduced into the Ghd7-deficient rice cv. Kita-ake and transgenic positive plants were checked by PCR with the primers flanking the intron. Red rows represent the two amplified fragments in the transgenic positive plants. (D) Phenotypic characterization of transgenic positive plants under NLD conditions. (E) Comparison of flowering time between the Ghd7 transgenic and wild-type plants under NLD conditions. (F) Circadian expression of OsCOL10 in the wild-type plants and Ghd7 transgenic lines. (G-H) Diurnal expression of OsCOL10 in the wild-type and se5 mutants with the background of Nipponbare (Nip) (G) or Gang46B (G46B) (H) under LD conditions. Penultimate leaf blades were harvested at indicated time point from 30-day-old (SDs) (B) and 35-day-old (LDs) (A, F, and GH) transgenic plants or mutants and corresponding wild-type plants. Open and filled bars at the bottom in (A-B) and (F-H) represent the light and dark periods, respectively. Expression levels are relative to the rice UBQ gene. Values are means ± sd of three independent biological replicates. sd, standard deviations.

228x309mm (300 x 300 DPI)



Figure 9. A proposed model of OsCOL10 in regulation of photoperiodic flowering time in rice. Blue lines represent pathways in LD and red lines indicate pathways shared between SD and LD. x, y and z represent existing but unknown gene(s). Dashed lines indicate unknown regulatory genes that may function by activation or repression. 147x272mm (600 x 600 DPI)

GmFT4, a homolog of FLOWERING LOCUS T, is positively regulated by E1 and functions as a flowering repressor in soybean.

IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice.

CaAP2 transcription factor is a candidate gene for a flowering repressor and a candidate for controlling natural variation of flowering time in Capsicum annuum.

EGR1 Functions as a Potent Repressor of MEF2 Transcriptional Activity.

Arabidopsis MSI1 functions in photoperiodic flowering time control.

Ghd7-Like Genes in Vernalization-Mediated Repression of Grass Flowering.

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis.

BosR functions as a repressor of the ospAB operon in Borrelia burgdorferi.

PV.1 induced by FGF-Xbra functions as a repressor of neurogenesis in Xenopus embryos.

Correction: EGR1 functions as a potent repressor of MEF2 transcriptional activity.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis.

TcNPR3 from Theobroma cacao functions as a repressor of the pathogen defense response.

Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice.

Overexpression of PvPin1, a Bamboo Homolog of PIN1-Type Parvulin 1, Delays Flowering Time in Transgenic Arabidopsis and Rice.

Automated characterization of flowering dynamics in rice using field-acquired time-series RGB images.

Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants.

A quantitative and dynamic model of the Arabidopsis flowering time gene regulatory network.

The effects of the photoperiod-insensitive alleles, se13, hd1 and ghd7, on yield components in rice.

qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O. sativa.

Homodimerization of Ehd1 Is Required to Induce Flowering in Rice.

The rice F-box protein KISS ME DEADLY2 functions as a negative regulator of cytokinin signalling.

[Shifting of the flowering time in colchicum].

The evolution of flowering strategies in US weedy rice.

A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor.