Photoperiodic Flowering In Rice Research Articles

OsCRY2 and OsFBO10 co-regulate photomorphogenesis and photoperiodic flowering in indica rice

Cryptochromes (CRYs) are a class of photoreceptors that perceive blue/ultraviolet-A light of the visible spectrum to mediate a vast number of physiological responses in bacteria, fungi, animals and plants. In the present study, we have characterized OsCRY2 in a photoperiod sensitive indica variety, Basmati 370, by generating and analyzing overexpression (OE) and knock-down (KD) transgenic lines. The OsCRY2OE lines displayed dwarfism as shown in their reduced plant height and leaf length, attributed largely by an overall reduction in their cell size. The OsCRY2OE lines flowered significantly earlier and showed shorter and broader seeds with an overall reduced seed weight. The OsCRY2KD lines showed contrasting phenotypes, such as increased plant height and delayed flowering, however, decreased seed size and weight were also observed in the KD lines, along with reduced spikelet fertility and high seed shattering rate in mature panicles. Novel interactions were confirmed between OsCRY2 and members of ZEITLUPE family of blue/ultraviolet-A light photoreceptors, encoded by OsFBO8, OsFBO9 and OsFBO10 which are orthologous to ZEITLUPE (ZTL), LOV KELCH PROTEIN2 (LKP2) and FLAVIN BINDING, KELCH REPEAT F-BOX1 (FKF1), respectively, of Arabidopsis thaliana. Since FKF1 is known to play a role in regulating photoperiodic flowering, OsFBO10 was chosen for further studies. OsCRY2 and OsFBO10 interacted in the nucleus and cytoplasm of the cell and cross-regulated the expression of each other. They were also found to regulate the expression of several genes involved in photoperiodic flowering in rice. Both OsCRY2 and OsFBO10 played a positive role in photomorphogenic responses in different light conditions. The physical interaction of OsCRY2 with OsFBO10, their involvement in common physiological and developmental pathways and their cross-regulation of each other suggest that the two photoreceptors may regulate common developmental pathways in plants, either jointly or redundantly.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7

IntroductionCircadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. ObjectivesIn this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s–OsELF3-1–OsLUX complex directly regulates flowering-related genes to mediate rice heading. MethodsWe identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s–OsELF3-1–OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s–OsELF3-1–OsLUX) complex. ResultsThe circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s–OsELF3-1–OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. ConclusionOur findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s–OsELF3-1–OsLUX)–Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

Autophagy targets Hd1 for vacuolar degradation to regulate rice flowering

Flowering time (heading date) is a critical agronomic trait that determines the yield and regional adaptability of crops. Heading date 1 (Hd1) is a central regulator of photoperiodic flowering in rice (Oryza sativa). However, how the homeostasis of Hd1 protein is achieved is poorly understood. Here, we report that the nuclear autophagy pathway mediates Hd1 degradation in the dark to regulate flowering. Loss of autophagy function results in an accumulation of Hd1 and delays flowering under both short-day and long-day conditions. In the dark, nucleus-localized Hd1 is recognized as a substrate for autophagy and is subjected to vacuolar degradation via the autophagy protein OsATG8. The Hd1-OsATG8 interaction is required for autophagic degradation of Hd1 in the dark. Our study reveals a new mechanism by which Hd1 protein homeostasis is regulated by autophagy to control rice flowering. Our study also indicates that the regulation of flowering by autophagic degradation of Hd1 orthologs may have arisen over the course of mesangiosperm evolution, which would have increased their flexibility and adaptability to the environment by modulating flowering time.

LUX ARRHYTHMO Interacts With ELF3a and ELF4a to Coordinate Vegetative Growth and Photoperiodic Flowering in Rice.

The evening complex (EC) plays a critical role in photoperiod flowering in Arabidopsis. Nevertheless, the underlying functions of individual components and coordinate regulation mechanism of EC genes in rice flowering remain to be elucidated. Here, we characterized the critical role of LUX ARRHYTHMO (LUX) in photoperiod perception and coordinating vegetative growth and flowering in rice. Non-functional alleles of OsLUX extremely extended vegetative phase, leading to photoperiod-insensitive late flowering and great increase of grain yield. OsLUX displayed an obvious diurnal rhythm expression with the peak at dusk and promoted rice flowering via coordinating the expression of genes associated with the circadian clock and the output integrators of photoperiodic flowering. OsLUX combined with OsELF4a and OsELF3a or OsELF3b to form two ECs, of which the OsLUX-OsELF3a-OsELF4a was likely the dominant promoter for photoperiodic flowering. In addition, OsELF4a was also essential for promoting rice flowering. Unlike OsLUX, loss OsELF4a displayed a marginal influence under short-day (SD) condition, but markedly delayed flowering time under long-day (LD) condition. These results suggest that rice EC genes share the function of promoting flowering. This is agreement with the orthologs of SD plant, but opposite to the counterparts of LD species. Taken together, rice EC genes display similar but not identical function in photoperiodic flowering, probably through regulating gene expression cooperative and independent. These findings facilitate our understanding of photoperiodic flowering in plants, especially the SD crops.

High Ambient Temperatures Inhibit Ghd7-Mediated Flowering Repression in Rice.

The anticipation of changing seasons is crucial for reproduction in plants. Despite the broad cultivation area, the effects of ambient temperature on photoperiodic flowering are largely unknown in rice. Here, we first examined flowering time under four distinct conditions: short-day or long-day and high or low temperature, using cultivars, nearly isogenic lines, and mutants in rice. We also examined gene expression patterns of key flowering-time genes using the same lines under various conditions including temporal dynamics after light pulses. In addition to delayed flowering because of low growth rates, we found that photoperiodic flowering is clearly enhanced by both Hd1 and Ghd7 genes under low-temperature conditions in rice. We also revealed that PhyB can control Ghd7 repressor activity as a temperature sensor to inhibit Ehd1, Hd3a and RFT1 at lower temperatures, likely through a post-transcriptional regulation, despite inductive photoperiod conditions. Furthermore, we found that rapid reduction of Ghd7 messenger RNA (mRNA) under high-temperature conditions can lead to mRNA increase in a rice florigen gene, RFT1. Thus, multiple temperature-sensing mechanisms can affect photoperiodic flowering in rice. The rising of ambient temperatures in early summer likely contributes to the inhibition of Ghd7 repressor activity, resulting in the appropriate floral induction of rice in temperate climates.

Dual function of clock component OsLHY sets critical day length for photoperiodic flowering in rice.

SummaryCircadian clock, an endogenous time‐setting mechanism, allows plants to adapt to unstable photoperiod conditions and induces flowering with proper timing. In Arabidopsis, the central clock oscillator was formed by a series of interlocked transcriptional feedback loops, but little is known in rice so far. By MutMap technique, we identified the candidate gene OsLHY from a later flowering mutant lem1 and further confirmed it through genetic complementation, RNA interference knockdown, and CRISPR/Cas9‐knockout. Global transcriptome profiling and expression analyses revealed that OsLHY might be a vital circadian rhythm component. Interestingly, oslhy flowered later under ≥12 h day length but headed earlier under ≤11 h day length. qRT‐PCR results exhibited that OsLHY might function through OsGI‐Hd1 pathway. Subsequent one‐hybrid assays in yeast, DNA affinity purification qPCR, and electrophoretic mobility shift assays confirmed OsLHY could directly bind to the CBS element in OsGI promoter. Moreover, the critical day length (CDL) for function reversal of OsLHY in oslhy (11–12 h) was prolonged in the double mutant oslhy osgi (about 13.5 h), indicating that the CDL set by OsLHY was OsGI dependent. Additionally, the dual function of OsLHY entirely relied on Hd1, as the double mutant oslhy hd1 showed the same heading date with hd1 under about 11.5, 13.5, and 14 h day lengths. Together, OsLHY could fine‐tune the CDL by directly regulating OsGI, and Hd1 acts as the final effector of CDL downstream of OsLHY. Our study illustrates a new regulatory mechanism between the circadian clock and photoperiodic flowering.

Elucidation of molecular mechanism of photoperiodic flowering in rice and its application

Elucidation of molecular mechanism of photoperiodic flowering in rice and its application

Genomic adaptation of flowering‐time genes during the expansion of rice cultivation area

The diversification of flowering time in response to natural environments is critical for the spread of crops to diverse geographic regions. In contrast with recent advances in understanding the molecular basis of photoperiodic flowering in rice (Oryza sativa), little is known about how flowering-time diversification is structured within rice subspecies. By analyzing genome sequencing data and a set of 429 chromosome segment substitution lines (CSSLs) originating from 10 diverse rice accessions with wide distributions, we revealed diverse effects of allelic variations for common flowering-time quantitative trait loci in the recipient's background. Although functional variations associated with a few loci corresponded to standing variations among subspecies, the identified functional nucleotide polymorphisms occurred recently after rice subgroup differentiation, indicating that the functional diversity of flowering-time gene sequences was not particularly associated with phylogenetic relationship between rice subspecies. Intensive analysis of the Hd1 genomic region identified the signature of an early introgression of the Hd1 with key mutation(s) in aus and temperate japonica accessions. Our data suggested that, after such key introgressions, new mutations were selected and accelerated the flowering-time diversity within subspecies during the expansion of rice cultivation area. This finding may imply that new genome-wide changes for flowering-time adaptation are one of the critical determinants for establishing genomic architecture of local rice subgroups. In-depth analyses of various rice genomes coupling with the genetically confirmed phenotypic changes in a large set of CSSLs enabled us to demonstrate how rice genome dynamics has coordinated with the adaptation of cultivated rice during the expansion of cultivation area.

A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice

Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.

The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice.

The photoperiodic response is one of the most important factors determining heading date in rice (Oryza sativa). Although rhythmic expression patterns of flowering time genes have been reported to fine-tune the photoperiodic response, posttranslational regulation of key flowering regulators has seldom been elucidated in rice. Heading date 1 (Hd1) encodes a zinc finger transcription factor that plays a crucial role in the photoperiodic response, which determines rice regional adaptability. However, little is known about the molecular mechanisms of Hd1 accumulation during the photoperiod response. Here, we identify a C3HC4 RING domain-containing E3 ubiquitin ligase, Heading date Associated Factor 1 (HAF1), which physically interacts with Hd1. HAF1 mediates ubiquitination and targets Hd1 for degradation via the 26S proteasome-dependent pathway. The haf1 mutant exhibits a later flowering heading date under both short-day and long-day conditions. In addition, the haf1 hd1 double mutant headed as late as hd1 plants under short-day conditions but exhibited a heading date similar to haf1 under long-day conditions, thus indicating that HAF1 may determine heading date mainly through Hd1 under short-day conditions. Moreover, high levels of Hd1 accumulate in haf1. Our results suggest that HAF1 is essential to precise modulation of the timing of Hd1 accumulation during the photoperiod response in rice.

Ds9 was isolated encoding as OsHAP3H and its C-terminus was required for interaction with HAP2 and HAP5

Heading date is one of the most important agronomic traits in rice. Here, we analyzed an early flowering mutant ds9 under long day (LD) conditions and found that the early flowering phenotype was controlled by a single dominant gene. Mapping the Ds9 revealed that it was located near SSR marker RM5432 on chromosome 8, close to the known heading date-related DTH8/Ghd8 locus. Sequence analysis indicated that Ds9 was allelic to Ghd8, encoding the OsHAP3H subunit of CCAAT-box binding proteins (HAPs), and transformation of ds9 mutant with the OsHAP3H gene from wild type XS11 plants significantly increased the heading date under LD conditions. Yeast two hybrid and three hybrid assays revealed that the C-terminus of OsHAP3H is required for its interactions with OsHAP2 and OsHAP5. The data demonstrated that OsHAP3H plays a critical role as a repressor of photoperiodic flowering in rice.

Ehd4 Encodes a Novel and Oryza-Genus-Specific Regulator of Photoperiodic Flowering in Rice

Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the “florigen” genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.

OsELF3-1, an Ortholog of Arabidopsis EARLY FLOWERING 3, Regulates Rice Circadian Rhythm and Photoperiodic Flowering

Arabidopsis thaliana EARLY FLOWERING 3 (ELF3) as a zeitnehmer (time taker) is responsible for generation of circadian rhythm and regulation of photoperiodic flowering. There are two orthologs (OsELF3-1 and OsELF3-2) of ELF3 in rice (Oryza sativa), but their roles have not yet been fully identified. Here, we performed a functional characterization of OsELF3-1 and revealed it plays a more predominant role than OsELF3-2 in rice heading. Our results suggest OsELF3-1 can affect rice circadian systems via positive regulation of OsLHY expression and negative regulation of OsPRR1, OsPRR37, OsPRR73 and OsPRR95 expression. In addition, OsELF3-1 is involved in blue light signaling by activating EARLY HEADING DATE 1 (Ehd1) expression to promote rice flowering under short-day (SD) conditions. Moreover, OsELF3-1 suppresses a flowering repressor GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (Ghd7) to indirectly accelerate flowering under long-day (LD) conditions. Taken together, our results indicate OsELF3-1 is essential for circadian regulation and photoperiodic flowering in rice.

Molecular Dissection of the Roles of Phytochrome in Photoperiodic Flowering in Rice

Phytochromes mediate the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. Recent molecular genetics studies have revealed a genetic network that enables the critical daylength response of florigen gene expression. Analyses using a rice phytochrome chromophore-deficient mutant, photoperiod sensitivity5, have so far revealed that within this network, phytochromes are required for expression of Grain number, plant height and heading date7 (Ghd7), a floral repressor gene in rice. There are three phytochrome genes in rice, but the roles of each phytochrome family member in daylength response have not previously been defined. Here, we revealed multiple action points for each phytochrome in the critical daylength response of florigen expression by using single and double phytochrome mutant lines of rice. Our results show that either phyA alone or a genetic combination of phyB and phyC can induce Ghd7 mRNA, whereas phyB alone causes some reduction in levels of Ghd7 mRNA. Moreover, phyB and phyA can affect Ghd7 activity and Early heading date1 (a floral inducer) activity in the network, respectively. Therefore, each phytochrome gene of rice has distinct roles, and all of the phytochrome actions coordinately control the critical daylength response of florigen expression in rice.

Ehd3, encoding a plant homeodomain finger‐containing protein, is a critical promoter of rice flowering

Oryza sativa (rice) flowers in response to photoperiod, and is a facultative short-day (SD) plant. Under SD conditions, flowering is promoted through the activation of FT-like genes (rice florigens) by Heading date 1 (Hd1, a rice CONSTANS homolog) and Early heading date 1 (Ehd1, with no ortholog in the Arabidopsis genome). On the other hand, under long-day (LD) conditions, flowering is delayed by the repressive function of Hd1 on FT-like genes and by downregulation of Ehd1 by the flowering repressor Ghd7 - a unique pathway in rice. We report here that an early heading date 3 (ehd3) mutant flowered later than wild-type plants, particularly under LD conditions, regardless of the Hd1-deficient background. Map-based cloning revealed that Ehd3 encodes a nuclear protein that contains a putative transcriptional regulator with two plant homeodomain (PHD) finger motifs. To identify the role of Ehd3 within the gene regulatory network for rice flowering, we compared the transcript levels of genes related to rice flowering in wild-type plants and ehd3 mutants. Increased transcription of Ghd7 under LD conditions and reduced transcription of downstream Ehd1 and FT-like genes in the ehd3 mutants suggested that Ehd3 normally functions as an LD downregulator of Ghd7 in floral induction. Furthermore, Ehd3 ghd7 plants flowered earlier and show higher Ehd1 transcript levels than ehd3 ghd7 plants, suggesting a Ghd7-independent role of Ehd3 in the upregulation of Ehd1. Our results demonstrate that the PHD-finger gene Ehd3 acts as a promoter in the unique genetic pathway responsible for photoperiodic flowering in rice.

Genetic and molecular dissection of flowering time in rice

Flowering time (heading date) is a major determinant of regional and seasonal adaptation of cultivated rice. A large amount of variation is observed in heading date and photoperiodic response among rice cultivars and strains, including wild relatives. Quantitative trait locus (QTL) analyses of progeny derived from several cross combinations of rice cultivars suggest that more than 15 loci are involved in heading date. Map-based cloning has been performed on several QTLs for photoperiodic response. We have demonstrated that Heading date 1 (Hd1) is an ortholog of CONSTANS (CO) in Arabidopsis and is involved in the promotion of heading under short-day (SD) conditions and inhibition under long-day (LD) conditions. Hd6 is involved in inhibition under LD conditions and encodes the alpha-subunit of protein kinase CK2. Hd3a shows a high level of similarity to T Early heading date 1 (Ehd1) is involved in promotion under SD conditions and encodes a B-type response regulator. Hd5 is involved in inhibition under LD conditions and encodes a putative subunit of a CCAAT-box-binding protein. Late heading date 4 (Lhd4) is involved in inhibition under LD conditions and encodes a protein with a CCT motif. The combining of information from genetic and sequencing analyses reveals that the combination of natural alleles with loss or gain of function at particular QTLs, such as Hd1, Hd5, Hd6, Ehd1, and Lhd4, seems to generate a wide range of continuous variation in photoperiodic flowering in rice. These genetic and molecular analyses have allowed us to propose a pathway for the genetic control of photoperiodic flowering in rice, and analysis of the mRNA levels of genes in near-isogenic lines has clearly revealed their hierarchical relationship in the genetic control pathway. Identification and expression analyses of genes suggest the conservation and divergence of various features in the photoperiodic control of flowering in rice, an SD plant, and Arabidopsis, an LD plant.

Analysis of PHOTOPERIOD SENSITIVITY5 Sheds Light on the Role of Phytochromes in Photoperiodic Flowering in Rice

A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a gamma-irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways.

Ehd2, a Rice Ortholog of the Maize INDETERMINATE1 Gene, Promotes Flowering by Up-Regulating Ehd1

Recent research into the flowering of rice (Oryza sativa) has revealed both unique and conserved genetic pathways in the photoperiodic control of flowering compared with those in Arabidopsis (Arabidopsis thaliana). We discovered an early heading date2 (ehd2) mutant that shows extremely late flowering under both short- and long-day conditions in line with a background deficient in Heading date1 (Hd1), a rice CONSTANS ortholog that belongs to the conserved pathway. This phenotype in the ehd2 mutants suggests that Ehd2 is pivotal for the floral transition in rice. Map-based cloning revealed that Ehd2 encodes a putative transcription factor with zinc finger motifs orthologous to the INDETERMINATE1 (ID1) gene, which promotes flowering in maize (Zea mays). Ehd2 mRNA in rice tissues accumulated most abundantly in developing leaves, but was present at very low levels around the shoot apex and in roots, patterns that are similar to those of ID1. To assign the position of Ehd2 within the flowering pathway of rice, we compared transcript levels of previously isolated flowering-time genes, such as Ehd1, a member of the unique pathway, Hd3a, and Rice FT-like1 (RFT1; rice florigens), between the wild-type plants and the ehd2 mutants. Severely reduced expression of these genes in ehd2 under both short- and long-day conditions suggests that Ehd2 acts as a flowering promoter mainly by up-regulating Ehd1 and by up-regulating the downstream Hd3a and RFT1 genes in the unique genetic network of photoperiodic flowering in rice.

OsCO3, a CONSTANS-LIKE gene, controls flowering by negatively regulating the expression of FT-like genes under SD conditions in rice

The photoperiod is an important environmental stress that determines flowering time. The CONSTANS (CO) and Heading date 1 (Hd1) genes are known to be central integrators of the photoperiod pathway in Arabidopsis and rice, respectively. Although they are both members of the CONSTANS-LIKE (COL) family and have two B-boxes and a CCT domain, rice also possesses novel COL genes that are not found in Arabidopsis. Here, we demonstrate that a novel COL gene, OsCO3, containing a single B-box and a CCT domain, modulates photoperiodic flowering in rice. The circadian expression pattern of OsCO3 mRNA oscillated in a different phase from Hd1 and was similar to that of OsCO3 pre-mRNA, suggesting that the diurnal expression pattern of OsCO3 transcripts may be regulated at the transcriptional level. Overexpression of OsCO3 specifically caused late flowering under short day (SD) conditions relative to wild-type rice plants. The expression of Hd3a and FTL decreased in these transgenic plants, whereas the expression of Hd1, Early heading date 1 (Ehd1), OsMADS51, and OsMADS50 did not significantly change. Our results suggest that OsCO3 primarily controls flowering time under SD conditions by negatively regulating Hd3a and FTL expression, independent of the SD-promotion pathway.