EARLY FLOWERING3 Research Articles

Heat Makes Cellular Hotspots in Plants

Temperature-sensitive phase separation is reversibly and rapidly controlled by the prion domain (PrD)-containing EARLY FLOWERING 3 (ELF3) protein. Disordered protein-driven liquid-liquid phase separation underlies a ratio-metric thermosensing in plants.

A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.

The diverse and unanticipated roles of histone deacetylase 9 in coordinating plant development and environmental acclimation.

Plants tightly control gene transcription to adapt to environmental conditions and steer growth and development. Different types of epigenetic modifications are instrumental in these processes. In recent years, an important role for the chromatin-modifying RPD3/HDA1 class I HDAC HISTONE DEACETYLASE 9 (HDA9) emerged in the regulation of a multitude of plant traits and responses. HDACs are widely considered transcriptional repressors and are typically part of multiprotein complexes containing co-repressors, DNA, and histone-binding proteins. By catalyzing the removal of acetyl groups from lysine residues of histone protein tails, HDA9 negatively controls gene expression in many cases, in concert with interacting proteins such as POWERDRESS (PWR), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15), WRKY53, ELONGATED HYPOCOTYL 5 (HY5), ABA INSENSITIVE 4 (ABI4), and EARLY FLOWERING 3 (ELF3). However, HDA9 activity has also been directly linked to transcriptional activation. In addition, following the recent breakthrough discovery of mutual negative feedback regulation between HDA9 and its interacting WRKY-domain transcription factor WRKY53, swift progress in gaining understanding of the biology of HDA9 is expected. In this review, we summarize knowledge on this intriguing versatile-and long under-rated-protein and propose novel leads to further unravel HDA9-governed molecular networks underlying plant development and environmental biology.

A mobile ELF4 delivers circadian temperature information from shoots to roots.

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Elucidating how the plant circadian clock responds to temperature oscillations is crucial to understand plant responsiveness to the environment. Here we found a prevalent temperature-dependent function of the Arabidopsis clock component ELF4 (EARLY FLOWERING 4) in the root clock. Although the root clock is able to run in the absence of shoots, micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information but trafficking is essential to control the period of the root clock in a temperature-dependent manner. At low temperatures, ELF4 mobility is favored, resulting in a slow-paced root clock while high temperatures decrease movement, leading to a faster clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.

ELF in the root.

Each plant organ or tissue senses a different type of environmental signal as input to the circadian clock and shares that information in some way. The small protein EARLY FLOWERING 4 (ELF4) functions as a shoot-to-root mobile signal, thereby allowing regulation of the root clock’s pace depending on ambient temperature.

Differential Effects of Day/Night Cues and the Circadian Clock on the Barley Transcriptome

The circadian clock is a complex transcriptional network that regulates gene expression in anticipation of the day/night cycle and controls agronomic traits in plants. However, in crops, how the internal clock and day/night cues affect the transcriptome remains poorly understood. We analyzed the diel and circadian leaf transcriptomes in the barley (Hordeum vulgare) cultivar 'Bowman' and derived introgression lines harboring mutations in EARLY FLOWERING3 (ELF3), LUX ARRHYTHMO1 (LUX1), and EARLY MATURITY7 (EAM7). The elf3 and lux1 mutants exhibited abolished circadian transcriptome oscillations under constant conditions, whereas eam7 maintained oscillations of ≈30% of the circadian transcriptome. However, day/night cues fully restored transcript oscillations in all three mutants and thus compensated for a disrupted oscillator in the arrhythmic barley clock mutants elf3 and lux1 Nevertheless, elf3, but not lux1, affected the phase of the diel oscillating transcriptome and thus the integration of external cues into the clock. Using dynamical modeling, we predicted a structure of the barley circadian oscillator and interactions of its individual components with day/night cues. Our findings provide a valuable resource for exploring the function and output targets of the circadian clock and for further investigations into the diel and circadian control of the barley transcriptome.

The Soybean Gene J Contributes to Salt Stress Tolerance by Up-Regulating Salt-Responsive Genes.

Soybean [Glycine max (L.) Merr.] is an important crop for oil and protein resources worldwide, and its farming is impacted by increasing soil salinity levels. In Arabidopsis the gene EARLY FLOWERING 3 (ELF3), increased salt tolerance by suppressing salt stress response pathways. J is the ortholog of AtELF3 in soybean, and loss-of-function J-alleles greatly prolong soybean maturity and enhance grain yield. The exact role of J in abiotic stress response in soybean, however, remains unclear. In this study, we showed that J expression was induced by NaCl treatment and that the J protein was located in the nucleus. Compared to NIL-J, tolerance to NaCl was significantly lower in the NIL-j mutant. We also demonstrated that overexpression of J increased NaCl tolerance in transgenic soybean hairy roots. J positively regulated expression of downstream salt stress response genes, including GmWRKY12, GmWRKY27, GmWRKY54, GmNAC, and GmSIN1. Our study disclosed a mechanism in soybean for regulation of the salt stress response. Manipulation of these genes should facilitate improvements in salt tolerance in soybean.

Molecular mechanisms of Evening Complex activity in Arabidopsis

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Primary seed dormancy is acquired during seed development and maturation, which is important for plant fitness and survival. DELAY OF GERMINATION1 (DOG1) plays a critical role in inducing seed dormancy. DOG1 expression increases rapidly during seed development, but the precise mechanism underlying this process remains elusive. In this study, we showed that mutants with a loss or reduced function of the chromatin-remodeling factor PICKLE (PKL) exhibit increased seed dormancy. PKL associates with DOG1 chromatin and inhibits its transcription. We found that PKL physically interacts with LUX ARRHYTHMO (LUX), a member of the evening complex (EC) of the circadian clock. Furthermore, LUX directly binds to a specific coding sequence of DOG1, and DOG1 acts genetically downstream of PKL and LUX. Mutations in either LUX or EARLY FLOWERING3 (ELF3) encoding another member of the EC led to increased DOG1 expression and enhanced seed dormancy. Surprisingly, these phenotypes were abolished when the parent plants were grown under continuous light. In addition, we observed that loss of function of either PKL or LUX decreased H3K27me3 levels at the DOG1 locus. Taken together, our study reveals a regulatory mechanism in which EC proteins coordinate with PKL to transmit circadian signals for directly regulating DOG1 expression and seed dormancy during seed development.

Evidence for rice Heading date 16 contribution to yield increase under low-nutrient conditions

ABSTRACT Breeding new rice cultivars that are tolerant to low-nutrient condition is a solution to saving the usage of commercial fertilizer. We isolated Hitomebore EMS mutant, 520-6. Seedling growth of 520-6 is tolerant to low-nutrient supply, especially to low phosphorus (low P) supply and genetic mapping revealed the existence of nonsynonymous substitution within Os03g0793500 9th exon in 520-6. Os03g0793500 is reported as Heading date 16 (Hd16)/Early flowering1 (EL1), which is related with the rice flowering pathway. Hd16/EL1 encodes a casein kinase-I protein, and phosphorylates the floral repressor Ghd7 and the rice DELLA protein Slender rice 1 (SLR1). 520-6 carries the same nonfunctional Hd16 allele to that of Koshihikari, and the parental cultivar, Hitomebore, carries the functional Hd16 allele, as with Nipponbare. mRNA accumulation of GA-biosynthesis-related gene, OsGA2ox3 was enhanced in 520-6 compared with Hitomebore, indicating that Hd16 takes a part in feedback regulation of the GA biosynthetic gene. 520-6 and near-isogenic line (NIL) with nonfunctional Hd16 allele showed heavy straw weight and panicle weight in paddy fields with low nitrogen input and no fertilizer application in Kashimadai at Miyagi prefecture and Hikone at Shiga prefecture, respectively. In addition, plants with nonfunctional Hd16 allele showed late flowering phenotype in all tested fields. These results imply that disruption of Hd16 contributes to the improvement of rice growth by positively regulating GA signaling and late flowering, which resulted in increased rice yield under low-nutrient conditions.

Heterologous expression of ELF4 from Chlamydomonas reinhardtii and Physcomitrella patens delays flowering in Arabidopsis thaliana

The circadian clock perceives seasonal changes in day length and temperature and then regulates numerous internal cellular processes. Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is a key circadian clock component that is involved in transcriptional regulation of the central oscillator and multiple output pathways. However, the functions of ELF4 orthologues in other organisms are poorly understood. Here, we identified ELF4 orthologous genes in two non-flowering organisms, the unicellular green alga Chlamydomonas (CrELF4) and the moss Physcomitrella patens (PpELF4). Constitutive expression of CrELF4 and PpELF4 in the Arabidopsis elf4 mutant not only completely rescued its early flowering phenotype, but also induced late flowering. This was associated with decreased transcript levels of CONSTANS (CO) and FLOWERING LOCUS T (FT), two key positive regulators of flowering-time pathways. In addition, expression of CrELF4 and PpELF4 in the Arabidopsis elf4 mutant inhibited hypocotyl elongation and altered the expression of circadian clock-related genes. Taken together, these data suggest that ELF4 orthologous genes in Chlamydomonas and Physcomitrella share an evolutionary conserved role with Arabidopsis ELF4 and indicate that the plant clock component ELF4 is functionally conserved from basal plants to angiosperms.

The EC-HDA9 complex rhythmically regulates histone acetylation at the TOC1 promoter in Arabidopsis

Circadian clocks are conserved time-keeper mechanisms in some prokaryotes and higher eukaryotes. Chromatin modification is emerging as key regulatory mechanism for refining core clock gene expression. Rhythmic changes in histone marks are closely associated to the TIMING OF CAB EXPRESSION 1 (TOC1) Arabidopsis clock gene. However, the chromatin-related modifiers responsible for these marks remain largely unknown. Here, we uncover that the chromatin modifier HISTONE DEACETYLASE 9 (HDA9) and the Evening complex (EC) component EARLY FLOWERING 3 (ELF3) directly interact to regulate the declining phase of TOC1 after its peak expression. We found that HDA9 specifically binds to the TOC1 promoter through the interaction with ELF3. The EC-HDA9 complex promotes H3 deacetylation at the TOC1 locus, contributing to suppressing TOC1 expression during the night, the time of EC function. Therefore, we have identified the mechanism by which the circadian clock intertwines with chromatin-related components to shape the circadian waveforms of gene expression in Arabidopsis.

CENTRORADIALIS Interacts with FLOWERING LOCUS T-Like Genes to Control Floret Development and Grain Number.

CENTRORADIALIS (CEN) is a key regulator of flowering time and inflorescence architecture in plants. Natural variation in the barley (Hordeum vulgare) homolog HvCEN is important for agricultural range expansion of barley cultivation, but its effects on shoot and spike architecture and consequently yield have not yet been characterized. Here, we evaluated 23 independent hvcen, also termed mat-c, mutants to determine the pleiotropic effects of HvCEN on developmental timing and shoot and spike morphologies of barley under outdoor and controlled conditions. All hvcen mutants flowered early and showed a reduction in spikelet number per spike, tiller number, and yield in the outdoor experiments. Mutations in hvcen accelerated spikelet initiation and reduced axillary bud number in a photoperiod-independent manner but promoted floret development only under long days (LDs). The analysis of a flowering locus t3 (hvft3) hvcen double mutant showed that HvCEN interacts with HvFT3 to control spikelet initiation. Furthermore, early flowering3 (hvelf3) hvcen double mutants with high HvFT1 expression levels under short days suggested that HvCEN interacts with HvFT1 to repress floral development. Global transcriptome profiling in developing shoot apices and inflorescences of mutant and wild-type plants revealed that HvCEN controlled transcripts involved in chromatin remodeling activities, cytokinin and cell cycle regulation and cellular respiration under LDs and short days, whereas HvCEN affected floral homeotic genes only under LDs. Understanding the stage and organ-specific functions of HvCEN and downstream molecular networks will allow the manipulation of different shoot and spike traits and thereby yield.

Arabidopsis ELF4-like proteins EFL1 and EFL3 influence flowering time

The circadian clock synchronizes internal and external stimuli to ensure numerous biological processes occur at the optimal time. EARLY FLOWERING 4 (ELF4) is a key evening-phased component of the circadian clock and essential for photoperiod-dependent flowering time regulation in Arabidopsis thaliana. There are four homologous ELF4-like (EFL1-EFL4) genes in the Arabidopsis genome but their functions are unknown. Protein sequence alignment and phylogenetic analysis showed that these four EFL proteins contained an evolutionarily conserved domain, DUF1313, of unknown function. To investigate the physical roles of these genes in Arabidopsis, we overexpressed the four homologous EFL genes in the elf4 mutant background. Under both long-day (LD) and short-day (SD) conditions, overexpression of EFL1 not only completely rescued the early flowering phenotype of the elf4 mutant, but also delayed flowering. Overexpression of EFL2, however, failed to rescue this phenotype and overexpression of EFL3 partially rescued the early flowering phenotype. The transcription levels of the key flowering time regulation genes CONSTANS (CO) and FLOWERING LOCUS T (FT) were significantly decreased in the EFL1- and EFL3-overexpressing transgenic lines in a dose-dependent manner, compared with the elf4 mutant. These results suggest that EFL1 and EFL3 are involved in flowering time regulation in Arabidopsis.

Arabidopsis JMJD5/JMJ30 Acts Independently of LUX ARRHYTHMO Within the Plant Circadian Clock to Enable Temperature Compensation.

The circadian system ensures that plants respond appropriately to environmental change by predicting regular transitions that occur during diel cycles. In order to be most useful, the circadian system needs to be compensated against daily and seasonal changes in temperature that would otherwise alter the pace of this biological oscillator. We demonstrate that an evening-phased protein, the putative histone demethylase JMJD5, contributes to temperature compensation. JMJD5 is co-expressed with components of the Evening Complex, an agglomeration of proteins including EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHYMO (LUX), which also integrates temperature changes into the molecular clockwork. One role of the Evening Complex is to regulate expression of PSEUDORESPONSE REGULATOR9 (PRR9) and PRR7, important components of the temperature compensation mechanism. Surprisingly we find that LUX, but not other Evening Complex components, is dispensable for clock function at low temperatures. Further genetic analysis suggests JMJD5 acts in a parallel pathway to LUX within the circadian system. Although an intact JMJD5 catalytic domain is required for its function within the clock, our findings suggest JMJD5 does not directly regulate H3K36 methylation at circadian loci. Such data refine our understanding of how JMDJ5 acts within the Arabidopsis circadian system.

HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA.

In plants, seasonal inputs such as photoperiod and temperature modulate the plant's internal genetic program to regulate the timing of the developmental transition from vegetative to reproductive growth. This regulation of the floral transition involves chromatin remodeling, including covalent modification of histones. Here, we report that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), a WD40 repeat protein, associates with a histone deacetylase complex to repress transcription of the GIGANTEA (GI)-mediated photoperiodic flowering pathway in Arabidopsis (Arabidopsis thaliana). Loss of function of HOS15 confers early flowering under long-day conditions because elevated GI expression. LUX ARRHYTHMO (LUX), a DNA binding transcription factor and component of the Evening Complex (EC), is important for the binding of HOS15 to the GI promoter. In wild type, HOS15 associates with the EC components LUX, EARLY FLOWERING 3 (ELF3), and ELF4 and the histone deacetylase HDA9 at the GI promoter, resulting in histone deacetylation and reduced GI expression. In the hos15-2 mutant, the levels of histone acetylation are elevated at the GI promoter, resulting in increased GI expression. Our data suggest that the HOS15-EC-HDA9 histone-modifying complex regulates photoperiodic flowering via the transcriptional repression of GI.

Genetic Relationship Between Phytochromes and OsELF3-1 Reveals the Mode of Regulation for the Suppression of Phytochrome Signaling in Rice.

EARLY FLOWERING3 (ELF3) functions as a night-time repressor required for sustaining circadian rhythms and co-ordinating growth and development in various plant species. The rice genome carries two ELF3 homologs, namely OsELF3-1 and OsELF3-2. Previous studies have suggested that OsELF3-1 has a predominant role in controlling rice photoperiodic flowering, while also contributing to the transcriptional regulation of rice floral regulators expressed in the morning. However, OsELF3-1 has not been functionally characterized. Here, we observed that the oself3-1 mutation suppresses the photoperiod-insensitive early flowering of photoperiod sensitivity5 (se5), which is a chromophore-deficient rice mutant. Detailed analyses of the se5oself3-1 double mutant revealed the recovery of the phytochrome-dependent expression of Grain number, plant height, and heading date7 (Ghd7), a floral repressor, and Light-harvesting chlorophyll a/b binding protein (Lhcb) genes. Although the oself3-1 mutation recovered Ghd7 expression in the se5 background, there was a lack of Ghd7 expression in the phyAphyBphyC triple mutant background. These observations suggest that OsELF3-1 represses Ghd7 expression by inhibiting the phytochrome signaling pathway. Comparative genome analyses indicated that OsELF3-1 was produced via gene duplication events in Oryza species, and that it is expressed throughout the day. A comparison between the oself3-1 mutant and transgenic rice lines in which OsELF3-1 and OsELF3-2 are simultaneously silenced uncovered a role for OsELF3-1 in addition to the canonical ELF3 function as an evolutionarily conserved role for a night-time repressor that regulates the rice circadian clock. Our study confirmed that an ELF3 paralog, OsELF3-1, had a unique role involving the suppression of phytochrome signaling.

Two B-Box Domain Proteins, BBX18 and BBX23, Interact with ELF3 and Regulate Thermomorphogenesis in Arabidopsis

Plants coordinate their growth and developmental programs with various endogenous signals and environmental challenges. Phytochrome interacting factor 4 (PIF4) plays a critical positive role in thermoresponsive gene expression and hypocotyl growth in Arabidopsis, whereas early flowering 3 (ELF3) negatively regulates the activity of PIF4 at elevated temperatures. However, it is unknown how ELF3 activity is regulated at warm temperatures. Here, we report the identification of B-box 18 (BBX18) and BBX23 as important thermomorphogenesis regulators in Arabidopsis. BBX18 and BBX23 mutations result in reduced thermoresponsive hypocotyl elongation. In contrast, BBX18 overexpression promotes hypocotyl growth at elevated temperatures, which depends oneither PIF4 or constitutive photomorphogenic 1 (COP1). BBX18 and BBX23 interact with ELF3 or COP1. Knocking out BBX18 and BBX23 increases ELF3 abundance under normal and warm temperature conditions. The expression of multiple thermoresponsive genes is impaired in both a PIF4 mutant and a BBX18/BBX23 double mutant. Thus, our findings reveal an important role of B-box proteins during thermomorphogenesis and provide insights into our understanding of how warm temperature signals regulate ELF3 activity and PIF4-dependent genes.

ClCRY2 facilitates floral transition in Chrysanthemum lavandulifolium by affecting the transcription of circadian clock-related genes under short-day photoperiods

Plants sense photoperiod signals to confirm the optimal flowering time. Previous studies have shown that Cryptochrome2 (CRY2) functions to promote floral transition in the long-day plant (LDP) Arabidopsis; however, the function and molecular mechanism by which CRY2 regulates floral transition in short-day plants (SDPs) is still unclear. In this study, we identified a CRY2 homologous gene, ClCRY2, from Chrysanthemum lavandulifolium, a typical SDP. The morphological changes in the C. lavandulifolium shoot apex and ClFTs expression analysis under SD conditions showed that adult C. lavandulifolium completed the developmental transition from vegetative growth to reproductive growth after eight SDs. Meanwhile, ClCRY2 mRNA exhibited an increasing trend from 0 to 8 d of SD treatment. ClCRY2 overexpression in wild-type (WT) Arabidopsis and C. lavandulifolium resulted in early flowering. The transcript levels of the CONSTANS-like (COL) genes ClCOL1, ClCOL4, and ClCOL5, and FLOWERING LOCUS T (FT) homologous gene ClFT1 were upregulated in ClCRY2 overexpression (ClCRY2-OE) C. lavandulifolium under SD conditions. The transcript levels of some circadian clock-related genes, including PSEUDO-REPONSE REGULATOR 5 (PRR5), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and GIGANTEA (GI-1 and GI-2), were upregulated in ClCRY2-OE C. lavandulifolium, while the expression levels of other circadian clock-related genes, such as EARLY FLOWERING 3 (ELF3), ELF4, LATE ELONGATED HYPOCOTYL (LHY), PRR73, and REVEILLE8 (RVE8), were downregulated in ClCRY2-OE C. lavandulifolium under SD conditions. Taken together, the results suggest that ClCRY2 promotes floral transition by fine-tuning the expression of circadian clock-related gene, ClCOLs and ClFT1 in C. lavandulifolium under SD conditions.

The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions.

The ubiquitin 26S proteasome system (UPS) is critical for enabling plants to alter their proteomes to integrate internal and external signals for the photoperiodic induction of flowering. We previously demonstrated that HAF1, a C3HC4 RING domain-containing E3 ubiquitin ligase, is essential to precisely modulate the timing of Heading Date1 accumulation and to ensure appropriate photoperiodic responses under short-day conditions in rice (Oryza sativa). However, how HAF1 mediates flowering under long-day conditions remains unknown. In this study, we show that OsELF3 (EARLY FLOWERING3) is the direct substrate of HAF1 for ubiquitination in vitro and in vivo. HAF1 is required for maintaining the circadian rhythm of OsELF3 accumulation during photoperiodic responses in rice. In addition, the haf1 oself3 double mutant headed as late as oself3 plants under long-day conditions. An amino acid variation (L558S) within the interaction domain of OsELF3 with HAF1 greatly contributes to the variation in heading date among japonica rice accessions. The japonica accessions carrying the OsELF3(L)-type allele are found at higher latitudes, while varieties carrying the OsELF3(S)-type allele are found at lower latitudes. Taken together, our findings suggest that HAF1 precisely modulates the diurnal rhythm of OsELF3 accumulation to ensure the appropriate heading date in rice.