Ef7 encodes an ELF3-like protein and promotes rice flowering by negatively regulating the floral repressor gene Ghd7 under both short- and long-day conditions

Much progress has been made in our understanding of photoperiodic flowering of rice and the mechanisms underlying short-day (SD) promotion and long-day (LD) repression of floral induction. In this study, we identified and characterized the Ef7 gene, one of the rice orthologs of Arabidopsis EARLY FLOWERING 3 (ELF3). The ef7 mutant HS276, which was induced by γ-irradiation of the japonica rice cultivar 'Gimbozu', flowers late under both SD and LD conditions. Expression analyses of flowering time-related genes demonstrated that Ef7 negatively regulates the expression of Ghd7, which is a repressor of the photoperiodic control of rice flowering, and consequently up-regulates the expression of the downstream Ehd1 and FT-like genes under both SD and LD conditions. Genetic analyses with a non-functional Ghd7 allele provided further evidence that the delayed flowering of ef7 is mediated through the Ghd7 pathway. The analysis of light-induced expression of Ghd7 revealed that the ef7 mutant was more sensitive to red light than the wild-type plant, but the gate of Ghd7 expression was unchanged. Thus, our results show that Ef7 functions as a floral promoter by repressing Ghd7 expression under both SD and LD conditions.     

The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.     

Circadian-associated rice pseudo response regulators (OsPRRs): insight into the control of flowering time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.     

CCA1 and ELF3 Interact in the control of hypocotyl length and flowering time in Arabidopsis

The circadian clock is an endogenous oscillator with a period of approximately 24 h that allows organisms to anticipate, and respond to, changes in the environment. In Arabidopsis (Arabidopsis thaliana), the circadian clock regulates a wide variety of physiological processes, including hypocotyl elongation and flowering time. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a central clock component, and CCA1 overexpression causes circadian dysfunction, elongated hypocotyls, and late flowering. EARLY FLOWERING3 (ELF3) modulates light input to the clock and is also postulated to be part of the clock mechanism. elf3 mutations cause light-dependent arrhythmicity, elongated hypocotyls, and early flowering. Although both genes affect similar processes, their relationship is not clear. Here, we show that CCA1 represses ELF3 by associating with its promoter, completing a CCA1-ELF3 negative feedback loop that places ELF3 within the oscillator. We also show that ELF3 acts downstream of CCA1, mediating the repression of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 in the control of hypocotyl elongation. In the regulation of flowering, our findings show that ELF3 and CCA1 either cooperate or act in parallel through the CONSTANS/FLOWERING LOCUS T pathway. In addition, we show that CCA1 represses GIGANTEA and SUPPRESSOR OF CONSTANS1 by direct interaction with their promoters, revealing additional connections between the circadian clock and the flowering pathways.     

Overexpression of Wheat <i>TaELF3-1BL</i> Delays Flowering in Arabidopsis

EARLY FLOWERING 3 (ELF3), a light zeitnehmer (time-taker) gene, regulates circadian rhythm and photoperiodic flowering in Arabidopsis, rice, and barley. The three orthologs of ELF3 (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) have been identified in wheat too, and one gene, TaELF3-1DL, has been associated with heading date. However, the basic characteristics of these three genes and the roles of the other two genes, TaELF3-1BL and, TaELF3-1AL, remain unknown. Therefore, the present study obtained the coding sequences of the three orthologs (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) of ELF3 from bread wheat and characterized them and investigated the role of TaELF3-1BL in Arabidopsis. Protein sequence comparison revealed similarities among the three TaELF3 genes of wheat; however, they were different from the Arabidopsis ELF3. Real-time quantitative PCR revealed TaELF3 expression in all wheat tissues tested, with the highest expression in young spikes; the three genes showed rhythmic expression patterns also. Furthermore, the overexpression of the TaELF3-1BL gene in Arabidopsis delayed flowering, indicating their importance in flowering. Subsequent overexpression of TaELF3-1BL in the Arabidopsis ELF3 nonfunctional mutant (elf3 mutant) eliminated its early flowering phenotype, and slightly delayed flowering. The wild-type Arabidopsis overexpressing TaELF3-1BL demonstrated reduced expression levels of flowering-related genes, such as CONSTANS (AtCO), FLOWERING LOCUS T (AtFT), and GIGANTEA (AtGI). Thus, the study characterized the three TaELF3 genes and associated TaELF3-1BL with flowering in Arabidopsis, suggesting a role in regulating flowering in wheat too. These findings provide a basis for further research on TaELF3 functions in wheat.     

Double loss-of-function mutation in EARLY FLOWERING 3 and CRYPTOCHROME 2 genes delays flowering under continuous light but accelerates it under long days and short days: an important role for Arabidopsis CRY2 to accelerate flowering time in continuous light

The photoperiodic response is one of the adaptation mechanisms to seasonal changes of lengths of day and night. The circadian clock plays pivotal roles in this process. In Arabidopsis, LHY, CCA1, ELF3, and other clock proteins play major roles in maintaining circadian rhythms. lhy;cca1 double mutants with severe defects in circadian rhythms showed accelerated flowering under short days (SDs), but delayed flowering under continuous light (LL). The protein level of the floral repressor SVP increased in lhy;cca1 mutants under LL, and the late-flowering phenotype of lhy;cca1 mutants was partially suppressed by svp, flc, or elf3. ELF3 interacted with both CCA1 and SVP, and elf3 suppressed the SVP accumulation in lhy;cca1 under LL. These results suggest that the unique mechanism of the inversion of the flowering response of lhy;cca1 under LL may involve both the ELF3-SVP/FLC-dependent and -independent pathways. In this work, elf3-1 seeds were mutagenized with heavy-ion beams and used to identify mutation(s) that delayed flowering under LL but not long days (LDs) or SDs even without ELF3. In this screening, seven candidate lines named suppressor of elf3 1 (self1), sel3, sel5, sel7, sel14, sel15, and sel20 were identified. Genetic analysis indicated that sel20 was a new deletion allele of a mutation in the blue light receptor, CRY2. A late-flowering phenotype and decrease of FT expression in the elf3;sel20 double mutant was obvious under LL but not under SDs or LDs. These results indicated that the late-flowering phenotype in the double mutant elf3;sel20 as well as in lhy;cca1 was affected by the presence of darkness. The results suggest that CRY2 may play more essential roles in the acceleration of flowering under LL than LDs or SDs.     

GIGANTEA and EARLY FLOWERING 4 in Arabidopsis exhibit differential phase-specific genetic influences over a diurnal cycle

The endogenous circadian clock regulates many physiological processes related to plant survival and adaptability. GIGANTEA (GI), a clock-associated protein, contributes to the maintenance of circadian period length and amplitude, and also regulates flowering time and hypocotyl growth in response to day length. Similarly, EARLY FLOWERING 4 (ELF4), another clock regulator, also contributes to these processes. However, little is known about either the genetic or molecular interactions between GI and ELF4 in Arabidopsis. In this study, we investigated the genetic interactions between GI and ELF4 in the regulation of circadian clock-controlled outputs. Our mutant analysis shows that GI is epistatic to ELF4 in flowering time determination, while ELF4 is epistatic to GI in hypocotyl growth regulation. Moreover, GI and ELF4 have a synergistic or additive effect on endogenous clock regulation. Gene expression profiling of gi, elf4, and gi elf4 mutants further established that GI and ELF4 have differentially dominant influences on circadian physiological outputs at dusk and dawn, respectively. This phasing of GI and ELF4 influences provides a potential means to achieve diversity in the regulation of circadian physiological outputs, including flowering time and hypocotyl growth.     

ELF3: a circadian safeguard to buffer effects of light.

The EARLY FLOWERING 3 (ELF3) gene of Arabidopsis regulates plant morphology, flowering time and circadian rhythms. ELF3 was proposed to function as a modulator of light signal transduction downstream of phytochromes, and, perhaps, other photoreceptors. Recent work indicates that ELF3 encodes a novel nuclear protein that is expressed rhythmically and interacts with phytochrome B. How ELF3 mediates the circadian gating of light responses and regulates light input to the clock is the subject of discussion.     

OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP ( http://rgp.dna.affrc.go.jp ). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3 ; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3 , which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation.     

Control of flowering in rice through synthetic microProteins

Photoperiod‐dependent flowering in rice is regulated by HEADING DATE 1 (Hd1), which acts as both an activator and repressor of flowering in a daylength‐dependent manner. To investigate the use of microProteins as a tool to modify rice sensitivity to the photoperiod, we designed a synthetic Hd1 microProtein (Hd1miP) capable of interacting with Hd1 protein, and overexpressed it in rice. Transgenic OX‐Hd1miP plants flowered significantly earlier than wild type plants when grown in non‐inductive long day conditions. Our results show the potential of microProteins to serve as powerful tools for modulating crop traits and unraveling protein function.     

An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15

In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering.     

Comparative overviews of clock-associated genes of Arabidopsis thaliana and Oryza sativa

In higher plants, circadian rhythms are highly relevant to a wide range of biological processes. To such circadian rhythms, the clock (oscillator) is central, and recent intensive studies on the model higher plant Arabidopsis thaliana have begun to shed light on the molecular mechanisms underlying the functions of the central clock. Such representative clock-associated genes of A. thaliana are the homologous CCA1 and LHY genes, and five PRR genes that belong to a small family of pseudo-response regulators including TOC1. Others are GI, ZTL, ELF3, ELF4, LUX/PCL1, etc. In this context, a simple question arose as to whether or not the molecular picture of the model Arabidopsis clock is conserved in other higher plants. Here we made an effort to answer the question with special reference to Oryza sativa, providing experimental evidence that this model monocot also has a set of highly conserved clock-associated genes, such as those designated as OsCCA1, OsPRR-series including OsTOC1/OsPRR1, OsZTLs, OsPCL1 as well as OsGI. These results will provide us with insight into the general roles of plant circadian clocks, such as those for the photoperiodic control of flowering time that has a strong impact on the reproduction and yield in many higher plants.