FLAVIN-BINDING KELCH REPEAT F-BOX1 Research Articles

Genome-wide identification of clock-associated genes and circadian rhythms in Fragaria × ananassa seedlings

Flowering time in plants is regulated by a photoperiod-responsive mechanism. Some plant species use a circadian clock-based control mechanism to adapt to variable environments. Strawberry is a horticultural crop that responds to certain photoperiods and temperatures to induce flowering. However, clock-associated genes in octoploid cultivated strawberry (Fragaria × ananassa) have not been defined, and their regulatory mechanism for responding to photoperiods is unclear. We herein targeted 12 clock-associated genes reported in other plant species and performed a genome-wide analysis and expression comparison in F. × ananassa seedlings. Seventy-eight sequences were selected from the F. × ananassa genome. The major domains and cis-acting elements were conserved in each sequence. Transcripts were clearly expressed under continuous light conditions in F. × ananassa seedlings (‘Yotsuboshi’) acclimated to long days. Among them, 9 genes maintained their unique autonomous circadian rhythms and may function as clock genes. LHY (LATE ELONGATED HYPOCOTYL) had the Myb domain and LHY expression peaked in the dawn. PRR (PSEUDO-RESPONSE REGULATOR) family members (PRR9, PRR7, PRR5, and TOC1 (TIMING OF CAB EXPRESSION 1)) had a pseudo-receiver domain and CCT domain, and peak expression times began sequentially from the afternoon for PRR9 to the evening for TOC1. LUX (LUXARRHYTHMO) had a Myb domain, and LUX expression peaked in evening with ELF3 (EARLY FLOWERING 3). FKF1 (FLAVIN-BINDING KELCH REPEAT F BOX 1) had PAS and F-box domains, and FKF1 expression peaked in the afternoon. GI (GIGANTEA) expression also peaked in the afternoon. F. × ananassa (‘Yotsuboshi’) appears to have multiple feedback loops comprising clock-associated genes. Although the rhythmic expression of CHE (CCA1 HIKING EXPEDITION) and ZTL (ZEITLUPE) was not observed, they had conserved domains, CHE with the TCP domain and ZTL with the PAS and F-box domains. The present results provide basic information on the circadian clock for the control of F. × ananassa flowering.

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known.

FKF1 F-box protein promotes flowering in part by negatively regulating DELLA protein stability under long-day photoperiod in Arabidopsis.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) encodes an F-box protein that regulates photoperiod flowering in Arabidopsis under long-day conditions (LDs). Gibberellin (GA) is also important for regulating flowering under LDs. However, how FKF1 and the GA pathway work in concert in regulating flowering is not fully understood. Here, we showed that the mutation of FKF1 could cause accumulation of DELLA proteins, which are crucial repressors in GA signaling pathway, thereby reducing plant sensitivity to GA in flowering. Both in vitro and in vivo biochemical analyses demonstrated that FKF1 directly interacted with DELLA proteins. Furthermore, we showed that FKF1 promoted ubiquitination and degradation of DELLA proteins. Analysis of genetic data revealed that FKF1 acted partially through DELLAs to regulate flowering under LDs. In addition, DELLAs exerted a negative feedback on FKF1 expression. Collectively, these findings demonstrate that FKF1 promotes flowering partially by negatively regulating DELLA protein stability under LDs, and suggesting a potential mechanism linking the FKF1 to the GA signaling DELLA proteins.

Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440-669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440-669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.

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.

Evolutionary conservation and functional divergence of the LFK gene family play important roles in the photoperiodic flowering pathway of land plants.

ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1)-blue-light photoreceptors-play important roles in regulating the circadian clock and photoperiodic flowering pathway in plants. In this study, phylogenetic analysis revealed that the LOV (Light, Oxygen, or Voltage) and Kelch repeat-containing F-box (LFK) gene family can be classified into two clades, ZTL/LKP2 and FKF1, with clear differentiation between monocots and dicots within each clade. The LFK family genes underwent strong purifying selection; however, signatures of positive selection to adapt to local conditions still existed in 18 specific codons. In 87 diverse maize inbred lines, significant differences were identified (P ≤ 0.01) for days to female flowering between the haplotypes consisting of eight positive selection sites at ZmFKF1b corresponding to tropical and temperate maize groups of the phylogenetic tree, indicating a key role of ZmFKF1b in maize adaptive evolution. In addition, positive coevolution was detected in the domains of the LFK family for long-term cooperation to targets. The Type-I and Type-II functional divergence analysis revealed subfunctionalization or neofunctionalization of the LFKs, and the ZTL subfamily is most likely to maintain the ancestral function of LFKs. Over 50% of critical amino acid sites involved in the functional divergence were identified in the Kelch repeat domain, resulting in the distinction of substrates for ubiquitination and degradation. These results suggest that evolutionary conservation contributes to the maintenance of critical physiological functions, whereas functional divergence after duplication helps to generate diverse molecular regulation mechanisms.

Arabidopsis TOE proteins convey a photoperiodic signal to antagonize CONSTANS and regulate flowering time.

Plants flower in an appropriate season to allow sufficient vegetative development and position flower development in favorable environments. In Arabidopsis, CONSTANS (CO) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) promote flowering by inducing FLOWER LOCUS T (FT) expression in the long-day afternoon. The CO protein is present in the morning but could not activate FT expression due to unknown negative mechanisms, which prevent premature flowering before the day length reaches a threshold. Here, we report that TARGET OF EAT1 (TOE1) and related proteins interact with the activation domain of CO and CO-like (COL) proteins and inhibit CO activity. TOE1 binds to the FT promoter near the CO-binding site, and reducing TOE function results in a morning peak of the FT mRNA. In addition, TOE1 interacts with the LOV domain of FKF1 and likely interferes with the FKF1-CO interaction, resulting in partial degradation of the CO protein in the afternoon to prevent premature flowering.

Evening expression of arabidopsis GIGANTEA is controlled by combinatorial interactions among evolutionarily conserved regulatory motifs.

Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarily conserved module present in GI and other evening-expressed genes.

Co-option of a photoperiodic growth-phase transition system during land plant evolution.

Photoperiodic control of the phase transition from vegetative to reproductive growth is critical for land plants. The GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) protein complex controls this process in angiosperms. However, little is known about how plants evolved this regulatory system. Here, we report that orthologues of GI and FKF1 are present in a basal plant, the liverwort Marchantia polymorpha, and describe the molecular interaction between their products. Knockout of either the GI or FKF1 orthologue completely abolishes the long-day-dependent growth-phase transition in M. polymorpha. Overexpression of either gene promotes growth-phase transition, even under short-day conditions. Introduction of the GI orthologue partially rescues the late-flowering phenotype of the Arabidopsis thaliana gi mutant. Our findings suggest that plants had already acquired the GI-FKF1 system to regulate growth-phase transition when they colonized land, and that this system was co-opted from gametophyte to sporophyte generation during evolution.

Diurnal expression of <i>CONSTANS-like</i> genes is independent of the function of cycling DOF factor (CDF)-like transcriptional repressors in <i>Physcomitrella patens</i>

The Dof-type transcriptional repressors CYCLING DOF FACTORS (CDFs) directly suppress the expression of CONSTANS (CO), which encodes a key regulator of photoperiodic gene expression, day-length perception and the floral transition in Arabidopsis thaliana. The genes encoding CDF-like (PpDof3 and PpDof4) and CO-like (PpCOL1-PpCOL3) proteins are present in the genome of the moss Physcomitrella patens, although P. patens lacks the genes encoding homologues of FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) and GIGANTEA (GI), which control the stability of CDFs in A. thaliana. In the current study, we investigated whether the functions of PpDof3 and PpDof4 are associated with the expression of PpCOL1-PpCOL3 in P. patens. We found that the diurnal expression patterns of PpDof3 and PpDof4 are similar to those of CDF genes and that like CDF1 from A. thaliana, PpDof3 and PpDof4 function as transcriptional repressors. However, targeted disruptions of PpDof3 and PpDof4 did not affect the expression of PpCOL1-PpCOL3, indicating that the expression of COLs is independent of the functions of PpDof3 and PpDof4 in P. patens.

Diverse Responses to Blue Light via LOV Photoreceptors

Plants utilize light not only as an energy source for photosynthesis, but also as an environmental cue to direct numerous developmental and physiological processes. For this purpose, plants have evolved two main types of photoreceptors: the phytochromes (Franklin and Quail 2010), which sense red/ far-red light, and the ultraviolet (UV)-A/blue light (BL)-sensing photoreceptors, which include cryptochrome (Liu et al. 2011), phototropin (Christie 2007) and other BL receptors described below. In addition, a third class of UV-B receptor, UVR8, was recently identified (Rizzini et al. 2011). The long history of research on BL receptors dates back to the initial observation made by the famous Charles Darwin, in which he found that plants were able to move towards light (Darwin and Darwin 1881). For a long time, the nature of the chromophore in BL receptors was much disputed as to whether it was a flavin or a carotenoid (Senger 1980). This issue was partially resolved through the identification of the first BL receptor, which corresponded to a flavin protein named cryptochrome (Ahmad and Cashmore 1993). Shortly after this discovery, the Briggs group demonstrated that the disrupted gene in Arabidopsis non-phototropic hypocotyl 1 (nph1) mutants encoded a BL receptor required for phototropic response (Huala et al. 1997, Christie et al. 1998). A homolog of NPH1, NON-PHOTOTROPIC HYPOCOTYL-LIKE 1 (NPL1), was soon identified (Kagawa et al. 2001); thereafter, NPH1 and NPL1 were renamed phototropin 1 (phot1) and phot2, respectively. Phototropins mediate diverse plant responses, such as chloroplast relocation movements (Kagawa et al. 2001, Sakai et al. 2001), stomatal opening (Kinoshita et al. 2001), early hypocotyl growth inhibition (Folta and Spaulding 2001), leaf flattening (Sakamoto and Briggs 2002), leaf positioning (Inoue et al. 2005), nuclear positioning (Iwabuchi et al. 2007, Tsuboi et al. 2007), sun tracking (Inoue et al. 2008b) and leaf photomorphogenesis (Kozuka et al. 2011), in which phot1 acts over a broad range of light intensities, whereas phot2 acts as a high-light sensor. These physiological responses contribute towards enhancing photosynthesis in the plant by increasing the absorption of light and CO2 (Takemiya et al. 2005). In addition to the two phototropins, a BL receptor family of proteins characteristically containing one light, oxygen or voltage (LOV) domain, an F-box and a Kelch repeat has been identified in Arabidopsis. These proteins include ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) and LOV KELCH PROTEIN 2 (LKP2), and are proposed to regulate the circadian clock and photoperiodic flowering by controlling BL-dependent protein degradation (Ito et al. 2012). A third type of LOV protein, aureochrome (AUREO), was identified in the stramenopile alga, Vaucheria frigida (Takahashi et al. 2007). AUREO has a basic region/leucine zipper (bZIP) domain as well as a LOV domain, and is thought to act as a BL-regulated transcription factor. Thus, the LOV BL receptor family in plants has divergent members who have a variety of physiological functions triggered by the photochemical reactions of FMN in the LOV domain.

LOV KELCH PROTEIN2 and ZEITLUPE repress Arabidopsis photoperiodic flowering under non‐inductive conditions, dependent on FLAVIN‐BINDING KELCH REPEAT F‐BOX1

LOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) constitute a family of Arabidopsis F-box proteins that regulate the circadian clock. Over-expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over-expressing LKP2, CO and FT expression was down-regulated under long-day conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Over-expression of LKP2 Kelch repeats induced late flowering under long-day conditions. lkp2 ztl double mutant plants flowered earlier than wild-type plants under short-day (non-inductive) conditions, and both CO and FT expression levels were up-regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non-inductive conditions, and this is dependent on FKF1.

Almost in a Day’s Work

Many plants coordinate their annual time of flowering with particular seasons of the year, and one indicator that they use to determine the change of seasons is day length. Sawa et al . (see the Perspective by Rubio and Deng) provide some insights into the molecular interactions that translate day length into flowering initiation. The day needs to be long enough for the blue-light stimulated expression of the proteins FKF1 (flavin-binding, kelch repeat, F-box1) and GIGANTIA (GI) to coordinate. The daily rise in expression of GI lags far enough behind the daily rise in expression of FKF1 that, in the shorter days of the year, daylight has waned by the time there is enough GI to form complexes with FKF1. With the longer days of spring and summer, there is enough time to form complexes and signal to the CONSTANS gene to trigger the flowering pathway. M. Sawa, D. A. Nusinow, S. A. Kay, T. Imaizumi, FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis . Science 318 , 261-265 (2007). [Abstract] [Full Text] V. Rubio, X. W. Deng, Standing on the shoulders of GIGANTEA. Science 318 , 206-207 (2007). [Summary] [Full Text]

Altered oscillator function affects clock resonance and is responsible for the reduced day‐length sensitivity of CKB4 overexpressing plants

Most organisms have evolved a timing mechanism or circadian clock that is able to generate 24 h rhythmic oscillations in multiple biological events. The environmental fluctuations in light and temperature synchronize the expression and activity of key oscillator components that ultimately define the period, phase and amplitude of output rhythms. In Arabidopsis, overexpression of the casein kinase 2 (CK2) regulatory subunits, CKB3 or CKB4, alters the function of the clock under free-running conditions, and results in period-shortening of genes peaking at different phase angles. Here, we examine the effects of CKB4 overexpression (CKB4-ox) on a number of clock outputs that are modulated by day length or photoperiod. We have found a phase shift in gene expression, shortening of hypocotyl elongation and earlier than wild-type initiation of flowering under short-day conditions. Our study shows that the earlier expression phases of the floral induction genes GIGANTEA, FLAVIN-BINDING KELCH REPEAT F-BOX1 and CONSTANS correlate with higher abundance of the FLOWERING LOCUS T transcript under short-day conditions. Matching the period of the external light/dark cycles relative to the endogenous short period of the CKB4-ox oscillator restores the phase of gene expression and the flowering sensitivity to day length, indicating that a clock defect is responsible for the CKB4-ox phenotypes. Our studies suggest a function for CKB4 very close to the oscillator, as expression of the core components TIMING OF CAB EXPRESSION 1 and CIRCADIAN CLOCK ASSOCIATED 1 is also altered in CKB4-ox plants. Based on our results, we propose that oscillator dysfunction is responsible for the period defect of CKB4-ox plants that leads to clock dissonance with the environment and reduced sensitivity to day length.

Conservation and divergence of circadian clock operation in a stress-inducible Crassulacean acid metabolism species reveals clock compensation against stress.

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator.