Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice

Yasuyuki Takahashi; Kosuke M. Teshima; Shuji Yokoi; Hideki Innan; Ko Shimamoto

doi:10.1073/pnas.0812092106

New Research In

Edited by Richard M. Amasino, University of Wisconsin, Madison, WI, and approved January 26, 2009 (received for review December 3, 2008)

Abstract

Rice is a facultative short-day plant, and molecular genetic studies have identified the major genes involved in short-day flowering. However, the molecular mechanisms promoting the diversity of flowering time in cultivated rice are not known. We used a core collection of 64 rice cultivars that represent the genetic diversity of 332 accessions from around the world and studied the expression levels and polymorphisms of 6 genes in the short-day flowering pathway. The RNA levels of Heading date 3a (Hd3a), encoding a floral activator, are highly correlated with flowering time, and there is a high degree of polymorphism in the Heading date 1 (Hd1) protein, which is a major regulator of Hd3a expression. Functional and nonfunctional alleles of Hd1 are associated with early and late flowering, respectively, suggesting that Hd1 is a major determinant of variation in flowering time of cultivated rice. We also found that the type of Hd3a promoter and the level of Ehd1 expression contribute to the diversity in flowering time and Hd3a expression level. We evaluated the contributions of these 3 factors by a statistical analysis using a simple linear model, and the results supported our experimental observations.

Keywords:

Rice (Oryza sativa L.) has evolved during the last 8,000 to 10,000 years of domestication and breeding (1, 2). A major reason for the spread of rice cultivation to a wide range of geographical regions, and for the increases in yield, is the diversification of flowering time (1). In general, rice is known as a short-day plant that induces transition from the vegetative phase to the reproductive phase when it senses a decrease in day length. The molecular genetic pathway for short-day flowering in cultivated rice (Fig. 1A) is relatively well characterized (3–5). Signals from light and circadian clocks are received by OsGI, the rice orthologue of Arabidopsis GIGANTEA, and it regulates expression of Heading date 1 (Hd1) and OsMADS51 (6–8). Hd1 and its Arabidopsis orthologue CONSTANS encode zinc-finger type transcriptional activators with the CO, CO-like, and TOC1 (CCT) domains (9). Hd1 regulates Heading date 3a (Hd3a) expression (7, 9, 10). Hd3a is a rice orthologue of Arabidopsis FLOWERING LOCUS T (FT), and these genes recently were shown to encode a mobile flowering signal (11–16). RICE FLOWERING LOCUS T1 (RFT1) belongs to the rice FT-like gene family and functions as a floral activator, acting redundantly with Hd3a (17, 18). OsMADS51 encodes a type I MADS-box gene and functions upstream of Early heading date 1 (Ehd1) (8). Ehd1 encodes a B-type response regulator and acts as an activator of Hd3a independently from Hd1 (19). No clear orthologues of Ehd1 or OsMADS51 are found in the Arabidopsis genome. Although the genetic pathway for short-day flowering in rice is relatively well understood, the molecular mechanisms generating the diversity of flowering time in cultivated rice are not known. In this study, we analyzed the expression and nucleotide sequences of genes involved in short-day flowering in rice. Our study revealed that allelic variation of Hd1 is a main source of flowering time diversity in cultivated rice. We also found that diversity in Ehd1 expression levels and Hd3a promoter sequences contributes to diversity in flowering time and the Hd3a expression level.

Results

Hd3a Expression Is Highly Correlated with Flowering Time in Cultivated Rice.

To analyze the molecular mechanisms controlling variation in flowering time of cultivated rice, we used a core collection of 64 cultivars (20). This collection was developed from an original set of 332 cultivars to study the molecular diversity present in cultivated rice, and it represents 91% of the genetic diversity present in the original cultivars, as determined by restriction fragment length polymorphism analysis. Flowering times of cultivars in the collection were highly diverse, ranging from 45 days to 153 days after sowing (Fig. 1B). To identify the genes that are the major determinants of this diversity, we first analyzed the mRNA levels of the 6 flowering time genes shown in Fig. 1A in the core collection cultivars and looked for possible relationships between their expression levels and flowering times. Expression levels of OsGI, Hd1, and OsMADS51 were uniform among the cultivars, and little correlation with flowering time was found (Fig. 1 C–E, I). In contrast, expression levels of Ehd1, Hd3a, and RFT1 were highly variable (Fig. 1 F–H). Among these genes, RNA levels of Hd3a were most closely correlated with flowering time (Fig. 1 C–H), indicating that higher Hd3a expression levels were associated with earlier flowering time. A similar correlation also was observed in a later stage of plant development [supporting information (SI) Fig. S1]. Expression levels of Ehd1 correlated moderately with flowering time (Fig. 1 F and I). These results suggest that mRNA levels of Hd3a strongly affect the flowering time of cultivated rice and are consistent with previous results showing that Hd3a is a major floral activator in rice (9, 7, 18). Functional associations between the genes were detected also (Fig. 1I). The expression of OsMADS51 was correlated with that of OsGI, consistent with the recent finding that OsMADS51 expression is regulated by OsGI (8). Moreover, Hd3a expression was correlated with Ehd1 expression, as shown previously (19). Thus, genetic variations that are able to create diversity in Hd3a mRNA levels are the major determinants of variation in flowering time in rice. Candidates for such factors are the regulatory region of Hd3a and variations in the functions of 2 genes that regulate Hd3a expression, Hd1 and Ehd1 (Fig. 1A).

Fig. 1.

Variation in flowering time and relationships between RNA levels of flowering time genes and flowering times among cultivated rice. (A) A schematic model of the short-day flowering pathway in rice. (B) Distribution of mean flowering time under short-day conditions (10 h light, 14 h dark) in the core collection. (C–H) Correlation of flowering time with RNA levels of (C) OsGI; (D) OsMADS51; (E) Hd1; (F) Ehd1; (G) Hd3a; (H) RFT1. RNA levels in leaves of 35-day-old plants were determined by real-time RT-PCR and are shown as natural logarithms. RSK, cv. Ryou Suisan Koumai. (I) Pearson's correlation coefficient between flowering time and gene expression level. ** indicates P < 0.001, and * indicates P < 0.01.

Nucleotide Polymorphisms in the Promoter and Coding Regions of Hd3a.

First, we investigated the diversity in the regulatory region of Hd3a among the cultivars in core collection. The 2-kbp region of the Hd3a promoter previously was shown to be sufficient for proper regulation of Hd3a expression (7, 12). Therefore, nucleotide polymorphisms were analyzed over a 2-kb region including the promoter and the 5′ UTR region, and 7 types of sequences were identified (Fig. 2A). They were classified into 2 groups according to their sequences: Types 1 and 2 in Group A and Types 3–7 in Group B (Fig. 2B). Genes with Group B promoters were expressed at significantly higher levels than those with Group A promoters, suggesting an association between promoter sequence types and expression levels (Fig. 2C). Thus, the Hd3a promoter sequence is a potential factor generating diversity in flowering time. We analyzed several potential cis-elements in the promoter. We found no nucleotide changes in the CCAAT box (21). The Golden2, Arabidopsis RESPONSE REGULATOR (ARR) and Chlamydomonas regulatory protein of P-starvation acclimatization response (Psr1) (GARP) DNA-binding domain of Ehd1 potentially can bind the Hd3a promoter region via the ARR1 binding element (19). Three potential ARR1 binding sites were found in the Hd3a promoter, but a nucleotide change was found in an element at −1932 bp. No changes in the other 2 ARR1 binding elements were detected (Fig. 2A). Thus, no major alterations in potential regulatory sequences of the Hd3a promoter were identified. An association between the promoter type and expression level (Fig. 2C) may be caused by other unidentified sequences or by other loci that are tightly linked with these polymorphic sites.

Fig. 2.

Functions of Hd3a, OsMADS51, and Ehd1 are conserved among cultivated rice. The nucleotide sequences of the 3 genes in the cultivars in the core collection were compared with those of Nipponbare. Polymorphic nucleotides are indicated by different colors. (A) Nucleotide polymorphisms in the Hd3a promoter region. Red arrows indicate ARR1 binding elements, the blue arrow indicates the CCAAT box, and the yellow arrow indicates the TATA box. Deletion and insertion sites are shown by open and closed arrowheads, respectively. A GA repeat sequence is indicated by gray arrowheads. The 5′-untranslated region is colored in red. (B) Phylogenetic tree of the Hd3a promoter created by the unweighted pair-group method using arithmetic average. Numbers indicate genetic distance. (C) Hd3a RNA levels in cultivars with Group A and Group B Hd3a promoters. RNA levels in leaves of 35-day-old plants were determined by real-time RT-PCR and are shown as natural logarithms. Error bars represent SD. (D) Nucleotide polymorphisms in the Hd3a coding sequence. Red lines indicate the locations of codons for amino acids that are essential for potential ligand binding pocket. The blue box indicates the external loop domain. (E) Nucleotide polymorphisms in the Ehd1 coding sequence. Asp (D₁, D₂) and Lys (K) residues are indicated by yellow lines. A single amino acid substitution in the GARP domain, found in cv. DANYU, is shown in red. (F) Nucleotide polymorphisms in the OsMADS 51 coding sequence. A deletion site is indicated by an open arrowhead.

We found 6 types of Hd3a coding sequences in the core collection (Fig. 2D). No variations were found in the amino acids surrounding the potential ligand binding pocket or the external loop domain (residues 130–143), which is proposed to be important for FT protein function (22) (Fig. 2D). Therefore, we concluded that the Hd3a protein function is highly conserved among rice cultivars in the core collection.

Nucleotide Polymorphisms in Ehd1 and OsMADS51.

We examined possible diversity in the Ehd1 coding region and found an amino acid substitution (G219R) in the GARP domain of a Chinese cultivar, DANYU (Type 2) (Fig. 2E). This Gly-to-Arg substitution was described previously and was shown to decrease the DNA binding activity of Ehd1 (19). In the receiver domain of Ehd1, 3 amino acid residues, D₁, D₂, and K, are known to be important for protein function (19, 23). In all the cultivars except DANYU, no amino acid substitutions that might affect protein function were found in either the receiver or the GARP domains. Thus, the Ehd1 protein function is highly conserved in cultivated rice.

Because mRNA levels of Ehd1 were correlated moderately with those of Hd3a (Fig. 1 F and I), genes functioning upstream of Ehd1 potentially could provide variations in Ehd1 expression. Therefore, we also analyzed nucleotide diversity in OsMADS51 and identified 3 allele types. We found 6 amino acid substitutions, 2 in the MADS domain and 4 in other regions of the protein. The Type 2 alleles also carried a frame-shift mutation at the C-terminal end (Fig. 2F). However, these allelic variations of OsMADS51 were not correlated with Ehd1 mRNA levels (Fig. S2). We also investigated nucleotide polymorphism in the Ehd1 promoter regions. Analysis of more than 2 kbp in the upstream regions identified 9 types of sequences (Fig. S3). We analyzed the relationship between the allelic variation of Ehd1 promoter regions and the Ehd1 expression levels using Scheffé's F test, but we observed no significant difference.

Nucleotide Polymorphism of Hd1.

In contrast to Ehd1 and OsMADS51, we identified a high degree polymorphism in the Hd1 sequences, some of which cause frame-shift mutations or create premature stop codons. The Hd1 alleles in the core collection were grouped into 17 types, and 15 distinct proteins were identified (Fig. 3A). In addition to the previously reported 2-bp deletion found in cultivar (cv.) Kasalath (Fig. 3A, Type 13) (9), we discovered 3 deletions and 1 SNP that could cause defects in the entire or part of the CCT domains of 8 protein types (Fig. 3B). The CCT domain functions as a nuclear localization signal, and the lack of the CCT domain in the Arabidopsis CO causes a defect in the protein function (24). Thus, the Hd1 proteins encoded by these 9 allele types are likely to be nonfunctional. Although all the other alleles contained intact zinc-finger and CCT domains, we found several SNPs and indels that could cause amino acid substitutions in other regions of the protein (Fig. 3A). Therefore, we determined whether these Hd1 proteins were functional using a previously described transient assay in rice protoplasts (7). Although Hd1 activates Hd3a promoter activity under short-day conditions in planta, overexpression of functional Hd1 suppresses Hd3a promoter activity in this transient assay (7). Protoplasts were transformed with the Hd3a promoter-luciferase construct along with 1 of the Hd1 alleles, linked to the cauliflower mosaic virus (CaMV) 35S promoter, and we evaluated Hd1 function by measuring the suppression level of luciferase activity. In the reporter assay, the putatively functional Hd1 alleles showed decreased luciferase activity when compared with the nonfunctional Hd1 alleles (Fig. 3C), suggesting that these 8 types of Hd1 alleles encode functional Hd1 proteins. In conclusion, we found that Hd1 has a much higher level of polymorphism than the other genes examined in this study (Tables S1 and S2).

Fig. 3.

A high degree of polymorphism in the Hd1 coding sequence and its relationship with flowering time. (A) The Hd1 nucleotide sequences of the cultivars in the core collection were compared with that of cv. Ginbouzu. Polymorphic nucleotides are indicated by different colors. Deletion and insertion sites are indicated by open and closed arrowheads, respectively. The number of cultivars with each type of sequence (Type 1–17) is shown in the column at the right, with the numbers for loss-of-function types in red. Types 1 and 4 are identical, and Types 15 and 17 are identical. F.S., frame shift. (B) Structures of loss-of-function Hd1 proteins that lack the entire or part of the CCT domain. The locations of amino acid substitutions that cause loss of protein function are shown by red arrowheads. The open and closed arrowheads indicate deletion and insertion sites, respectively. The numbers below the arrowheads indicate which sequence types have the substitutions. Gray boxes indicate altered amino acid sequences caused by frame shifts. *, premature stop codon. (C) Activities of Hd1 alleles, analyzed by transient luciferase reporter assays using rice protoplasts. Red and blue bars indicate functional and nonfunctional Hd1 alleles, respectively. The Bar gene was used as a vector control. Error bars represent SD; n = 12–16. (D) Correlation of flowering times with Hd3a RNA levels in cultivars that carry functional Hd1 alleles (red) and nonfunctional Hd1 alleles (blue). RNA levels in leaves of 35-day-old plants were determined by real-time RT-PCR and are shown as natural logarithms. a, cv. RYOU SUISAN KOUMAI; b, cv. KHAU TAN CHIEM; c, cv. KEMASIN; d, cv. BINGALA; e, NIPPONBARE; f, hd1 mutant (NIPPONBARE); g, cv. Taichung 65. (E) Geographical distribution of functional (red) and nonfunctional (blue) Hd1 alleles. Alleles that were not sequenced are shown by gray columns. The number within each bar indicates the number of cultivars belonging to each Hd1 allelic group.

Correlation of the Allelic Diversity in Hd1 and Flowering Time.

To determine whether the allelic diversity of Hd1 was correlated with variations in the flowering times of cultivated rice, we grouped all the cultivars into those with functional and nonfunctional Hd1 alleles and compared their Hd3a mRNA levels with flowering times (Fig. 3D). Cultivars that carry functional Hd1 alleles tended to show higher Hd3a expression levels and earlier flowering times, whereas those carrying nonfunctional Hd1 alleles tended to show lower Hd3a expression levels and later flowering times (Fig. 3D). The differences in Hd3a expression levels and flowering times between the functional and nonfunctional Hd1 alleles were statistically significant by a student's t test. These results suggest that the nucleotide polymorphisms observed in Hd1 might be one of the main causes for the diversity of flowering time in cultivated rice. Cultivars with functional and nonfunctional Hd1 alleles were mapped to various regions of Asia (Fig. 3E). Results indicate that both groups of alleles are widely distributed, indicating that they have spread to many regions during the last 8,000 to 10,000 years.

Other Factors Contributing to the Diversity in Flowering Time of Rice.

Although we found significant differences in Hd3a expression levels and flowering times between cultivars carrying functional and nonfunctional Hd1 alleles, we also detected variations in each group, suggesting that other factors also may contribute to diversity in Hd3a expression levels and flowering times. We found that Ehd1 expression levels showed a moderate correlation with Hd3a expression level (r = 0.462, P < 0.001), and this correlation was little affected by the allelic variations of Hd1 (Table S3). Ehd1 was shown to induce Hd3a expression independently from Hd1 (19). Therefore, variation in Ehd1 expression levels also may contribute to the diversity of Hd3a expression levels and flowering times. This idea also is supported by the distribution of cv. NIPPONBARE (Hd1, Ehd1), hd1 mutant (hd1, Ehd1), and cv. Taicung 65 (hd1, ehd1) in Fig. 3D.

Linear Model Analysis of Flowering Time and Hd3a Expression Level in Cultivated Rice.

The relative contribution of various genetic factors to the phenotype variation in flowering time was assessed by applying a simple linear model that incorporates 3 genetic factors: Hd1 functionality, Hd3a promoter type, and Ehd1 expression level. This analysis revealed that the independent contributions of these genetic factors and the interactions between them account for 26.6% of the entire variance. The relative contributions of Hd1, Hd3a, and Ehd1 comprise 44.8%, 16.7%, and 38.5% of the genetic variance, respectively, indicating that the largest single contributor to flowering time variation is the difference between functional and nonfunctional Hd1 alleles. When phenotype was assessed by the expression level of Hd3a, 44.8% of the phenotypic variance could be explained by the 3 genetic factors; their relative contributions were 24.7%, 48.3%, and 27.0%, respectively, suggesting that variation in the Hd3a promoter contributes significantly more than the other 2 factors.

These observed differences in the contribution of each component to flowering time and Hd3a expression levels may indicate that other factors, such as posttranscriptional regulation of Hd3a, also are involved in the generation of flowering time diversity. It should be noted that these statistical analyses were performed using only Indica cultivars (n = 45) because of the small sample size available for Japonica cultivars (n = 13). We did not pool Indica and Japonica, to avoid any potential bias resulting from the Indica-Japonica population structure.

Discussion

In this study, we identified 3 factors that generate diversity in flowering time. Moreover, the contributions of these factors were calculated based on linear model analysis. Although we observed a strong correlation between flowering time and Hd3a expression level, we found differences in the relative contributions of the 3 genetic factors (the functionality of Hd1, the promoter type of Hd3a, and the expression level of Ehd1) to flowering time and Hd3a expression level. A possible explanation of these differences is that the variation in Hd3a promoter type directly affects the variation in Hd3a expression levels, whereas Hd1 and the expression level of Ehd1 could affect flowering time through other unknown factors. RFT1, for example, an orthologue of Hd3a that has been shown to affect flowering in the absence of functional Hd3a, would be an attractive subject for further studies on variations in promoter and protein-coding sequences (18). The current results support the genetic model for flowering time regulation in rice (Fig. 1A).

Our results also indicate that other, currently unknown, factors influence flowering time variation in cultivated rice. Several indirect factors such as growth rate, edaphic condition preference, and temperature may be among these unexplained factors. Variation in the activity of Hd1 alleles might be a factor. We performed a luciferase reporter assay to evaluate the activity of each Hd1 protein (Fig. 3C). Although we were able to distinguish functional proteins from nonfunctional proteins, it was difficult to demonstrate accurately differences in activity among Hd1 proteins. Variation of Hd3a expression level in each Hd1 allele could not be explained by the results of the luciferase reporter assay (Fig. 3C, Fig. S4), perhaps because of the complex regulation of Hd3a expression in rice (7). Allelic variations in the OsMADS51 and Ehd1 promoter regions could not explain the variation in Ehd1 expression levels (Fig. 2C and Fig. S2 and S3). Recently, RID1/Ehd2/OsId1, a rice orthologue of the maize ID1, was reported as a regulator of Ehd1 (25–27). Maize ID1 is a transcription factor that can bind to the specific DNA sequence TTTTGTCG/C (28). However, this consensus sequence is not found in the Ehd1 promoter region, and whether RID1/Ehd2/OsId1 is a direct target of Ehd1 promoter remains to be studied. Factors functioning upstream of Ehd1 that could generate variations in Ehd1 mRNA levels need to be identified in future work.

We found some exceptions in the correlation between Hd3a expression and flowering time. In particular, the Chinese cv. Ryou Suisan Koumai (RSK) showed significantly low Hd3a expression, but it flowered very early (58.5 days) (Fig. 1G). We found that RFT1 expression in RSK was much higher than in other cultivars (Figs. 1H and Fig. S5), suggesting that RFT1 may complement the low-level Hd3a expression. It may be possible to discover novel genes for flowering time regulation in rice, by studying this cultivar. We also found several exceptions: KHAU TAN CHIEM, KEMASIN, and BINGARA have a functional Hd1 allele but show low Hd3a levels and a late-flowering phenotype. However, because we did not find any anomalous values in the Hd1 and Ehd1 expression levels, other unknown factors that affect regulation of Hd3a expression by Hd1 proteins may be involved.

Natural variation in flowering time has been studied extensively in Arabidopsis. These studies have demonstrated that the major determinants of flowering time diversity are present in the vernalization pathway, which is a period of cold temperature that is required to induce flowering. FLOWERING LOCUS C (FLC), a flowering suppressor gene encoding a MADS-box transcription factor, and FRIGIDA (FRI), a positive regulator of FLC transcription, are the major factors controlling natural variation in flowering time in Arabidopsis (29–36).

Unlike Arabidopsis, rice is a tropical crop and does not show a vernalization response. Interestingly, no convincing orthologues of FLC and FRI were found in the rice genome (37). Temperate cereals such as wheat and barley also show vernalization responses; however, the genes involved in these responses are not conserved between Arabidopsis and cereals (38). The lack of homologues of Arabidopsis FLC in cereals may be consistent with these observations. The existence of variations in the Ppd-H1 gene, involved in the photoperiod pathway, among European barley cultivars has been described (38), but variations in the CO/Hd1 orthologues TaCO and HvCO in wheat and barley have not been reported (39, 40). However, the molecular genetic pathways controlling flowering time in wheat and barley have not yet been elucidated clearly, and the major determinants of flowering time in these species remain to be identified. Nevertheless, it appears that crops and plant species can adopt unique strategies to generate variations in flowering time and that the strategy used is one of the most important characters in the life of plants.

Materials and Methods

Plant Materials and Growth Conditions.

The rice core collection and Tos17-induced mutant of Hd1 (NG6082, Nipponbare) were obtained from the National Institute of Agrobiological Science GenBank (20, 41). The plants were grown in climate chambers under short-day conditions with daily cycles of 10 h of light at 30 °C and 14 h of dark at 25 °C. Light was provided by fluorescent white light (400–700 nm, 100 μmol m⁻² s⁻¹) at 70% humidity. Flowering time was defined as the time when the first panicle appeared from the node. Four to 8 independent plants were used to score flowering time.

Gene Expression Analysis.

Leaves were harvested from plants grown under short-day conditions, 35 days after germination at the zeitgeber times (ZT) corresponding to the expression peak for each gene, as follows: ZT 0 (Hd3a and RFT), ZT 8 (OsGI, OsMADS51), ZT 16 (Hd1), and ZT 20 (Ehd1). Leaf tissues were ground in liquid nitrogen with the ShakeMaster Auto ver.2.0 (BioMedical Science Inc.). Total RNA was extracted using TRIzol (Invitrogen) and treated with DNase I (Invitrogen). cDNA (20 μl) was synthesized from 1 μg of total RNA using SuperScriptII Reverse Transcriptase (Invitrogen). One μl of cDNA was used for the real-time PCR performed with the SYBR Green PCR master mix (Applied Biosystems). Data were collected using the ABI PRISM 7000 sequence detection system. All expression levels were normalized by that of ubiquitin. Four to 8 independent plants were used for this assay. Displayed data represent means of 2 separate RNA extractions.

Sequence Analysis.

Intron and promoter regions of each gene were amplified using LA Taq or PrimeSTAR HS DNA polymerase (Takara) from genomic DNA, and PCR products were purified by the MinElute system (Qiagen). These purified PCR fragments were used as templates, and sequencing reactions were performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Primers used for this analysis are listed in Table S4. Data were collected using the ABI PRISM 3100 Genetic Analyzer. The data were analyzed using the GENETYX program ver.7 (Genetyx).

Constructs for Luciferase Reporter Assay.

The Hd1 coding sequences were cloned from the cultivars in core collection (Table S2). These fragments were amplified by PCR with PrimeSTAR HS DNA polymerase (Takara). Hd1 fragments were subcloned into pENTR/D-TOPO vector by using the Directional TOPO cloning kit (Invitrogen). Subsequently, the Hd1 insert of pENTR plasmids was transferred to CaMV 35S::Gateway cassette plasmid, which contained pUC12 backbone, by performing an LR recombination reaction (Invitrogen). The Hd3a promoter sequence was cloned from cv. NIPPONBARE. This fragment was subcloned to pUC12 with pLUC (Photinus pyralis) plasmid using Ligation-Convenience Kit (Nippon Gene). Primers used for this experiment are listed in Table S4.

Luciferase Reporter Assays.

Rice protoplasts (2 × 10⁵ cells) were prepared from rice Oc cell cultures and transformed with 1 μg of pHd3a::pLUC (Photinus pyralis) as a reporter and 5 μg of each Hd1 construct, containing the Hd1 coding sequence driven by the CaMV 35S promoter using PEG transfection method (42). For a vector control, the bialaphos-resistance gene (Bar) was used instead of the Hd1 coding sequence. After incubation for 12 h, luciferase activities were measured using a Luciferase Assay System (Promega). Luciferase luminescence of each Hd1 allele was normalized for sample protein level and evaluated as a proportion of that of the vector control.

Statistical Analysis.

Statistical analyses were performed using Microsoft Excel 2003 or Statcel 2 (OMS). Correlation between flowering time and gene expression level was examined by Pearson's correlation coefficient test. The statistical differences in flowering times and Hd3a expression levels between plants with functional and nonfunctional Hd1 alleles were determined by the 2-tailed student's t test, and differences with P < 0.01 were considered to be significant. The effect of each haplotype on flowering time and gene expression was examined by ANOVA or Kruskal-Wallis test. If these analyses were significant, post hoc pairwise comparisons (Tukey-Kramer test or Scheffé's F test) were conducted, with the level of statistical significance taken as P < 0.01.

Linear Model Analysis.

To evaluate the relative contribution of the allelic diversity of Hd1 to the phenotypic variation in flowering time, we applied a simple linear model in which the phenotype of an individual is assumed to be given by y = G + E + e, where e signifies biological and technical replicates, and E represents all other genetic and environmental factors. G includes genetic variation caused by the functionality of Hd1, the promoter type of Hd3a, the expression level of Ehd1, and their interactions. We confirmed that there is almost no genetic linkage disequilibrium (LD) among these 3 loci. Although Hd3 and Hd1 are located on the same chromosome, the average LD of SNPs between the 2 regions is r² = 0.03, which is even smaller than the average LD between different chromosomes (r² = 0.06). However, we assumed a model in which the 3 interact because they belong to the same pathway. The same linear function can be applied using the expression level of Hd3a as the phenotype y. The statistical analysis was performed using R software.

Acknowledgments

We thank K. Ebana (National Institute of Agrobiological Sciences) for rice core collection, R. Ishikawa (Nara Institute of Science and Technology) for technical advice, and members of our laboratory for comments on the manuscript and discussions. The work described is supported by Grants-in-Aid for Scientific Research on Priority Areas (Grants 10182102 and 19090013) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.S.). Y.T. was supported by a fellowship from the Japanese Society for the Promotion of Science.

Footnotes

²To whom correspondence should be addressed. E-mail: simamoto{at}bs.naist.jp
Author contributions: Y.T., S.Y., and K.S. designed research; Y.T. performed research; Y.T., K.M.T., H.I., and K.S. analyzed data; and Y.T., H.I., and K.S. wrote the paper.
↵¹Present address: Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0812092106/DCSupplemental.
Freely available online through the PNAS open access option.

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OpenUrl CrossRef PubMed
↵
1. Kobayashi Y,
2. Weigel D
(2007) Move on up, it's time for change—mobile signals controlling photoperiod-dependent flowering. Genes Dev 21:2371–2384.
OpenUrl Abstract/FREE Full Text
↵
1. Izawa T,
2. et al.
(2002) Phytochrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice. Genes Dev 16:2006–2020.
OpenUrl Abstract/FREE Full Text
↵
1. Komiya R,
2. et al.
(2008) Hd3a and RFT1 are essential for flowering in rice. Development 135:767–774.
OpenUrl Abstract/FREE Full Text
↵
1. Doi K,
2. 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 Dev 18:926–936.
OpenUrl Abstract/FREE Full Text
↵
1. Kojima Y,
2. et al.
(2005) Development of an RFLP-based rice diversity research set of germplasm. Breed Sci 55:431–440.
OpenUrl CrossRef
↵
1. Wenkel S,
2. et al.
(2006) CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18:2971–2984.
OpenUrl Abstract/FREE Full Text
↵
1. Ahn JH,
2. et al.
(2006) A divergent external loop confers antagonistic activity on floral regulators FT and TFL1. EMBO J 25:605–614.
OpenUrl Abstract/FREE Full Text
↵
1. Sakai H,
2. Aoyama T,
3. Oka A
(2000) Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators. Plant J 24:703–711.
OpenUrl CrossRef PubMed
↵
1. Robson F,
2. et al.
(2001) Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant J 28:619–631.
OpenUrl CrossRef PubMed
↵
1. Wu C,
2. et al.
(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 USA 105:12915–12920.
OpenUrl Abstract/FREE Full Text
↵
1. Matsubara K,
2. et al.
(2008) Ehd2, a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating Ehd1. Plant Physiol 148:1425–1435.
OpenUrl Abstract/FREE Full Text
↵
1. Park SJ,
2. et al.
(2008) Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod. Plant J 56:1018–1029.
OpenUrl CrossRef PubMed
↵
1. Kozaki A,
2. Hake S,
3. Colasanti J
(2004) The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties. Nucleic Acids Res 32:1710–1720.
OpenUrl Abstract/FREE Full Text
↵
1. Michaels SD,
2. Amasino RM
(1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11:949–956.
OpenUrl Abstract/FREE Full Text
↵
1. Johanson U,
2. et al.
(2000) Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290:344–347.
OpenUrl Abstract/FREE Full Text
↵
1. Stinchcombe JR,
2. et al.
(2004) A latitudinal cline in flowering time in Arabidopsis thaliana modulated by the flowering time gene FRIGIDA. Proc Natl Acad Sci USA 101:4712–4717.
OpenUrl Abstract/FREE Full Text
↵
1. Caicedo AL,
2. et al.
(2004) Epistatic interaction between Arabidopsis FRI and FLC flowering time genes generates a latitudinal cline in a life history trait. Proc Natl Acad Sci USA 101:15670–15675.
OpenUrl Abstract/FREE Full Text
↵
1. Koornneef M,
2. Alonso-Blanco C,
3. Vreugdenhil D
(2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu Rev Plant Biol 55:141–172.
OpenUrl CrossRef PubMed
↵
1. Shindo C,
2. et al.
(2005) Role of FRIGIDA and FLOWERING LOCUS C in determining variation in flowering time of Arabidopsis. Plant Physiol 138:1163–1173.
OpenUrl Abstract/FREE Full Text
↵
1. Lempe J,
2. et al.
(2005) Diversity of flowering responses in wild Arabidopsis thaliana strains. PLoS Genetics 1:109–118.
OpenUrl PubMed
↵
1. Toomajian C,
2. et al.
(2006) A nonparametric test reveals selection for rapid flowering in the Arabidopsis genome. PLoS Biol 4:e137.
OpenUrl CrossRef PubMed
↵
1. Tadege M,
2. et al.
(2003) Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice. Plant Biotechnol J 1:336–369.
OpenUrl
↵
1. Cockram J,
2. et al.
(2007) Control of flowering time in temperate cereals: Genes, domestication, and sustainable productivity. J Exp Bot 58:1231–1244.
OpenUrl Abstract/FREE Full Text
↵
1. Griffiths S,
2. Dunford RP,
3. Coupland G,
4. Laurie DA
(2003) The evolution of CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant Physiol 131:1855–1867.
OpenUrl Abstract/FREE Full Text
↵
1. Nemoto Y,
2. et al.
(2003) Characterization and functional analysis of three wheat genes with homology to the CONSTANS flowering time gene in transgenic rice. Plant J 36:82–93.
OpenUrl CrossRef PubMed
↵
1. Hirochika H
(2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4:118–122.
OpenUrl CrossRef PubMed
↵
1. Chen S,
2. et al.
(2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427.
OpenUrl CrossRef PubMed

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Proceedings of the National Academy of Sciences: 106 (11)

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Article Classifications

Biological Sciences
Plant Biology

[1] ↵
Khush GS
(1997) Origin, dispersal, cultivation and variation of rice. Plant Mol Biol 35:25–34.
OpenUrl CrossRef PubMed

[2] Khush GS

[3] ↵
Doebley JF,
Gaut BS,
Smith BD
(2006) The molecular genetics of crop domestication. Cell 127:1309–1321.
OpenUrl CrossRef PubMed

[4] Doebley JF,

[5] Gaut BS,

[6] Smith BD

[7] ↵
Hayama R,
Coupland G
(2004) The molecular basis of diversity in the photoperiodic flowering responses of Arabidopsis and rice. Plant Physiol 135:677–684.
OpenUrl FREE Full Text

[8] Hayama R,

[9] Coupland G

[10] ↵
Izawa T
(2007) Adaptation of flowering-time by natural and artificial selection in Arabidopsis and rice. J Exp Bot 58:3091–3097.
OpenUrl Abstract/FREE Full Text

[11] Izawa T

[12] ↵
Tsuji H,
Tamaki S,
Komiya R,
Shimamoto K
(2008) Florigen and the photoperiodic control of flowering in rice. Rice 1:25–35.
OpenUrl CrossRef

[13] Tsuji H,

[14] Tamaki S,

[15] Komiya R,

[16] Shimamoto K

[17] ↵
Hayama R,
Izawa T,
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.
OpenUrl Abstract/FREE Full Text

[18] Hayama R,

[19] Izawa T,

[20] Shimamoto K

[21] ↵
Hayama R,
et al.
(2003) Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature 422:719–722.
OpenUrl CrossRef PubMed

[22] Hayama R,

[23] et al.

[24] ↵
Kim SL,
et al.
(2007) OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a. Plant Physiol 145:1484–1494.
OpenUrl Abstract/FREE Full Text

[25] Kim SL,

[26] et al.

[27] ↵
Yano M,
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 12:2473–2484.
OpenUrl Abstract/FREE Full Text

[28] Yano M,

[29] et al.

[30] ↵
Kojima S,
et al.
(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.
OpenUrl Abstract/FREE Full Text

[31] Kojima S,

[32] et al.

[33] ↵
Corbesier L,
et al.
(2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316:1030–1033.
OpenUrl Abstract/FREE Full Text

[34] Corbesier L,

[35] et al.

[36] ↵
Tamaki S,
et al.
(2007) Hd3a protein is a mobile flowering signal in rice. Science 316:1033–1036.
OpenUrl Abstract/FREE Full Text

[37] Tamaki S,

[38] et al.

[39] ↵
Lin MK,
et al.
(2007) FLOWERING LOCUS T protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell 19:1488–1506.
OpenUrl Abstract/FREE Full Text

[40] Lin MK,

[41] et al.

[42] ↵
Jaeger KE,
Wigge PA
(2007) FT protein acts as a long-range signal in Arabidopsis. Curr Biol 17:1050–1054.
OpenUrl CrossRef PubMed

[43] Jaeger KE,

[44] Wigge PA

[45] ↵
Mathieu J,
Warthmann N,
Kuttner F,
Schmid M
(2007) Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr Biol 17:1055–1060.
OpenUrl CrossRef PubMed

[46] Mathieu J,

[47] Warthmann N,

[48] Kuttner F,

[49] Schmid M

[50] ↵
Kobayashi Y,
Weigel D
(2007) Move on up, it's time for change—mobile signals controlling photoperiod-dependent flowering. Genes Dev 21:2371–2384.
OpenUrl Abstract/FREE Full Text

[51] Kobayashi Y,

[52] Weigel D

[53] ↵
Izawa T,
et al.
(2002) Phytochrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice. Genes Dev 16:2006–2020.
OpenUrl Abstract/FREE Full Text

[54] Izawa T,

[55] et al.

[56] ↵
Komiya R,
et al.
(2008) Hd3a and RFT1 are essential for flowering in rice. Development 135:767–774.
OpenUrl Abstract/FREE Full Text

[57] Komiya R,

[58] et al.

[59] ↵
Doi K,
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 Dev 18:926–936.
OpenUrl Abstract/FREE Full Text

[60] Doi K,

[61] et al.

[62] ↵
Kojima Y,
et al.
(2005) Development of an RFLP-based rice diversity research set of germplasm. Breed Sci 55:431–440.
OpenUrl CrossRef

[63] Kojima Y,

[64] et al.

[65] ↵
Wenkel S,
et al.
(2006) CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18:2971–2984.
OpenUrl Abstract/FREE Full Text

[66] Wenkel S,

[67] et al.

[68] ↵
Ahn JH,
et al.
(2006) A divergent external loop confers antagonistic activity on floral regulators FT and TFL1. EMBO J 25:605–614.
OpenUrl Abstract/FREE Full Text

[69] Ahn JH,

[70] et al.

[71] ↵
Sakai H,
Aoyama T,
Oka A
(2000) Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators. Plant J 24:703–711.
OpenUrl CrossRef PubMed

[72] Sakai H,

[73] Aoyama T,

[74] Oka A

[75] ↵
Robson F,
et al.
(2001) Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant J 28:619–631.
OpenUrl CrossRef PubMed

[76] Robson F,

[77] et al.

[78] ↵
Wu C,
et al.
(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 USA 105:12915–12920.
OpenUrl Abstract/FREE Full Text

[79] Wu C,

[80] et al.

[81] ↵
Matsubara K,
et al.
(2008) Ehd2, a rice ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating Ehd1. Plant Physiol 148:1425–1435.
OpenUrl Abstract/FREE Full Text

[82] Matsubara K,

[83] et al.

[84] ↵
Park SJ,
et al.
(2008) Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod. Plant J 56:1018–1029.
OpenUrl CrossRef PubMed

[85] Park SJ,

[86] et al.

[87] ↵
Kozaki A,
Hake S,
Colasanti J
(2004) The maize ID1 flowering time regulator is a zinc finger protein with novel DNA binding properties. Nucleic Acids Res 32:1710–1720.
OpenUrl Abstract/FREE Full Text

[88] Kozaki A,

[89] Hake S,

[90] Colasanti J

[91] ↵
Michaels SD,
Amasino RM
(1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11:949–956.
OpenUrl Abstract/FREE Full Text

[92] Michaels SD,

[93] Amasino RM

[94] ↵
Johanson U,
et al.
(2000) Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290:344–347.
OpenUrl Abstract/FREE Full Text

[95] Johanson U,

[96] et al.

[97] ↵
Stinchcombe JR,
et al.
(2004) A latitudinal cline in flowering time in Arabidopsis thaliana modulated by the flowering time gene FRIGIDA. Proc Natl Acad Sci USA 101:4712–4717.
OpenUrl Abstract/FREE Full Text

[98] Stinchcombe JR,

[99] et al.

[100] ↵
Caicedo AL,
et al.
(2004) Epistatic interaction between Arabidopsis FRI and FLC flowering time genes generates a latitudinal cline in a life history trait. Proc Natl Acad Sci USA 101:15670–15675.
OpenUrl Abstract/FREE Full Text

[101] Caicedo AL,

[102] et al.

[103] ↵
Koornneef M,
Alonso-Blanco C,
Vreugdenhil D
(2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu Rev Plant Biol 55:141–172.
OpenUrl CrossRef PubMed

[104] Koornneef M,

[105] Alonso-Blanco C,

[106] Vreugdenhil D

[107] ↵
Shindo C,
et al.
(2005) Role of FRIGIDA and FLOWERING LOCUS C in determining variation in flowering time of Arabidopsis. Plant Physiol 138:1163–1173.
OpenUrl Abstract/FREE Full Text

[108] Shindo C,

[109] et al.

[110] ↵
Lempe J,
et al.
(2005) Diversity of flowering responses in wild Arabidopsis thaliana strains. PLoS Genetics 1:109–118.
OpenUrl PubMed

[111] Lempe J,

[112] et al.

[113] ↵
Toomajian C,
et al.
(2006) A nonparametric test reveals selection for rapid flowering in the Arabidopsis genome. PLoS Biol 4:e137.
OpenUrl CrossRef PubMed

[114] Toomajian C,

[115] et al.

[116] ↵
Tadege M,
et al.
(2003) Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice. Plant Biotechnol J 1:336–369.
OpenUrl

[117] Tadege M,

[118] et al.

[119] ↵
Cockram J,
et al.
(2007) Control of flowering time in temperate cereals: Genes, domestication, and sustainable productivity. J Exp Bot 58:1231–1244.
OpenUrl Abstract/FREE Full Text

[120] Cockram J,

[121] et al.

[122] ↵
Griffiths S,
Dunford RP,
Coupland G,
Laurie DA
(2003) The evolution of CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant Physiol 131:1855–1867.
OpenUrl Abstract/FREE Full Text

[123] Griffiths S,

[124] Dunford RP,

[125] Coupland G,

[126] Laurie DA

[127] ↵
Nemoto Y,
et al.
(2003) Characterization and functional analysis of three wheat genes with homology to the CONSTANS flowering time gene in transgenic rice. Plant J 36:82–93.
OpenUrl CrossRef PubMed

[128] Nemoto Y,

[129] et al.

[130] ↵
Hirochika H
(2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4:118–122.
OpenUrl CrossRef PubMed

[131] Hirochika H

[132] ↵
Chen S,
et al.
(2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427.
OpenUrl CrossRef PubMed

[133] Chen S,

[134] et al.

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Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice

Abstract

Results

Hd3a Expression Is Highly Correlated with Flowering Time in Cultivated Rice.

Nucleotide Polymorphisms in the Promoter and Coding Regions of Hd3a.

Nucleotide Polymorphisms in Ehd1 and OsMADS51.

Nucleotide Polymorphism of Hd1.

Correlation of the Allelic Diversity in Hd1 and Flowering Time.

Other Factors Contributing to the Diversity in Flowering Time of Rice.

Linear Model Analysis of Flowering Time and Hd3a Expression Level in Cultivated Rice.

Discussion

Materials and Methods

Plant Materials and Growth Conditions.

Gene Expression Analysis.

Sequence Analysis.

Constructs for Luciferase Reporter Assay.

Luciferase Reporter Assays.

Statistical Analysis.

Linear Model Analysis.

Acknowledgments

Footnotes

References

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Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice

Abstract

Results

Hd3a Expression Is Highly Correlated with Flowering Time in Cultivated Rice.

Nucleotide Polymorphisms in the Promoter and Coding Regions of Hd3a.

Nucleotide Polymorphisms in Ehd1 and OsMADS51.

Nucleotide Polymorphism of Hd1.

Correlation of the Allelic Diversity in Hd1 and Flowering Time.

Other Factors Contributing to the Diversity in Flowering Time of Rice.

Linear Model Analysis of Flowering Time and Hd3a Expression Level in Cultivated Rice.

Discussion

Materials and Methods

Plant Materials and Growth Conditions.

Gene Expression Analysis.

Sequence Analysis.

Constructs for Luciferase Reporter Assay.

Luciferase Reporter Assays.

Statistical Analysis.

Linear Model Analysis.

Acknowledgments

Footnotes

References

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