Young Hun Song

FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering

Spring Into Flower In spring, plants respond to increasing day length and shifts in the spectrum of solar irradiance by releasing the flowering induction pathway, which includes expression of the FLOWERING LOCUS T (FT) protein. Song et al. (p. 1045) have now identified a trio of controls brought to bear on FT gene expression by the FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) protein. FKF1 removes a repressor and also stabilizes the activating CONSTANS (CO) protein in the afternoons through a binding interaction enhanced by blue light—an increasing component of solar irradiation during spring. Then FKF1 itself helps to activate transcription of the CO gene. Thus, by removing the repressor and shoring up the activator, FKF1 sets flowering on its way. A plant protein sensitive to blue light links longer afternoons to more flowering. Plants use day-length information to coordinate flowering time with the appropriate season to maximize reproduction. In Arabidopsis, the long day–specific expression of CONSTANS (CO) protein is crucial for flowering induction. Although light signaling regulates CO protein stability, the mechanism by which CO is stabilized in the long-day afternoon has remained elusive. Here, we demonstrate that FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) protein stabilizes CO protein in the afternoon in long days. FKF1 interacts with CO through its LOV domain, and blue light enhances this interaction. In addition, FKF1 simultaneously removes CYCLING DOF FACTOR 1 (CDF1), which represses CO and FLOWERING LOCUS T (FT) transcription. Together with CO transcriptional regulation, FKF1 protein controls robust FT mRNA induction through multiple feedforward mechanisms that accurately control flowering timing.

Flowering time regulation: photoperiod- and temperature-sensing in leaves

Plants monitor changes in photoperiod and temperature to synchronize their flowering with seasonal changes to maximize fitness. In the Arabidopsis photoperiodic flowering pathway, the circadian clock-regulated components, such as FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 and CONSTANS, both of which have light-controlled functions, are crucial to induce the day-length specific expression of the FLOWERING LOCUS T (FT) gene in leaves. Recent advances indicate that FT transcriptional regulation is central for integrating the information derived from other important internal and external factors, such as developmental age, amount of gibberellic acid, and the ambient temperature. In this review, we describe how these factors interactively regulate the expression of FT, the main component of florigen, in leaves.

F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control Arabidopsis clock progression.

In Arabidopsis thaliana, the F-box protein ZTL affects the period length of the circadian clock by regulating the stability of the core clock proteins TOC1 and PRR5. This study shows that, together with ZTL, the ZTL homologs FKF1 and LKP2 are also involved in the same protein stability regulation to determine the pace and robustness of the plant circadian clock. Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.

Photoperiodic Flowering: Time Measurement Mechanisms in Leaves

Many plants use information about changing day length (photoperiod) to align their flowering time with seasonal changes to increase reproductive success. A mechanism for photoperiodic time measurement is present in leaves, and the day-length-specific induction of the FLOWERING LOCUS T (FT) gene, which encodes florigen, is a major final output of the pathway. Here, we summarize the current understanding of the molecular mechanisms by which photoperiodic information is perceived in order to trigger FT expression in Arabidopsis as well as in the primary cereals wheat, barley, and rice. In these plants, the differences in photoperiod are measured by interactions between circadian-clock-regulated components, such as CONSTANS (CO), and light signaling. The interactions happen under certain day-length conditions, as previously predicted by the external coincidence model. In these plants, the coincidence mechanisms are governed by multilayered regulation with numerous conserved as well as unique regulatory components, highlighting the breadth of photoperiodic regulation across plant species.

Similarities in the circadian clock and photoperiodism in plants

Plants utilize circadian clocks to synchronize their physiological and developmental events with daily and yearly changes in the environment. Recent advances in Arabidopsis research have provided a better understanding of the molecular mechanisms of the circadian clock and photoperiodism. One of the most important questions is whether the mechanisms discovered in Arabidopsis are conserved in other plant species. Through the identification of many Arabidopsis clock gene homologs and the characterization of some gene functions, a strong resemblance between the circadian clocks in plants has been observed. On the contrary, based on our recent increased knowledge of photoperiodic flowering mechanisms in cereals and other plants, the day-length sensing mechanisms appear to have diverged more between long-day plants and short-day plants.

FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis

Many plants monitor day-length changes throughout the year and use the information to precisely regulate the timing of seasonal flowering for maximum reproductive success. In Arabidopsis thaliana, transcriptional regulation of the CONSTANS (CO) gene and posttranslational regulation of CO protein are crucial mechanisms for proper day-length measurement in photoperiodic flowering. Currently, the CYCLING DOF FACTOR proteins are the only transcription factors known to directly regulate CO gene expression, and the mechanisms that directly activate CO transcription have remained unknown. Here we report the identification of four CO transcriptional activators, named FLOWERING BHLH 1 (FBH1), FBH2, FBH3, and FBH4. All FBH proteins are related basic helix–loop–helix-type transcription factors that preferentially bind to the E-box cis-elements in the CO promoter. Overexpression of all FBH genes drastically elevated CO levels and caused early flowering regardless of photoperiod, whereas CO levels were reduced in the fbh quadruple mutants. In addition, FBH1 is expressed in the vascular tissue and bound near the transcription start site of the CO promoter in vivo. Furthermore, FBH homologs in poplar and rice induced CO expression in Arabidopsis. These results indicate that FBH proteins positively regulate CO transcription for photoperiodic flowering and that this mechanism may be conserved in diverse plant species. Our results suggest that the diurnal CO expression pattern is generated by a concert of redundant functions of positive and negative transcriptional regulators.

LOV Domain-Containing F-Box Proteins: Light-Dependent Protein Degradation Modules in Arabidopsis

Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, cryptochromes, and phototropins, ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains: a blue-light-absorbing LOV (Light, Oxygen, or Voltage) domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.

Distinct roles of FKF1, Gigantea, and Zeitlupe proteins in the regulation of Constans stability in Arabidopsis photoperiodic flowering.

Significance In many plants, seasonal flowering is a crucial aspect of maximizing reproductive fitness. Changes in day length (or photoperiod) provide the most reliable cue that enables plants to anticipate approaching seasonal variation in the surrounding environment. The induction of the FLOWERING LOCUS T (FT) gene controlled by the light-stabilized CONSTANS (CO) protein is the key mechanism for photoperiodic flowering in Arabidopsis thaliana. The CO/FT module is highly conserved in many plant species, including major crops. In Arabidopsis, blue-light–signaling pathways are essential for the stabilization of CO protein. Here we show a unique role of GIGANTEA in photoperiodic flowering through the interactions with two homologous proteins, FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and ZEITLUPE, which antagonistically control CO stability. Many plants measure changes in day length to synchronize their flowering time with appropriate seasons for maximum reproductive success. In Arabidopsis, the day-length–dependent regulation of CONSTANS (CO) protein stability is crucial to induce FLOWERING LOCUS T (FT) expression for flowering in long days. The FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) protein binds to CO protein specifically in the long-day afternoon and stabilizes it, although the mechanism remains unknown. Here we demonstrated that the FKF1-interacting proteins GIGANTEA (GI) and ZEITLUPE (ZTL) are involved in CO stability regulation. First, our immunoprecipitation-mass spectrometry analysis of FKF1 revealed that FKF1 forms an S-phase kinase-associated protein 1 (Skp1)/Cullin(CUL)/F-box complex through interactions with Arabidopsis Skp1-like 1 (ASK1), ASK2, and CUL1 proteins and mainly interacts with GI protein in vivo. GI interacts with CO directly and indirectly through FKF1. Unexpectedly, the gi mutation increases the CO protein levels in the morning in long days. This gi-dependent destabilization of CO protein was cancelled by the fkf1 mutation. These results suggest that there are other factors likely influenced by both gi and fkf1 mutations that also control CO stability. We found that ZTL, which interacts with GI and FKF1, may be one such factor. ZTL also interacts with CO in vivo. The CO protein profile in the ztl mutant resembles that in the gi mutant, indicating that ZTL activity also may be changed in the gi mutant. Our findings suggest the presence of balanced regulation among FKF1, GI, and ZTL on CO stability regulation for the precise control of flowering time.

Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature

Clock‐regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best‐understood clock output pathways in Arabidopsis, which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to CYCLING DOF FACTOR 1 (CDF1) and FLAVIN‐BINDING, KELCH REPEAT, F‐BOX 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed‐forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock‐regulated transcription of PHYTOCHROME‐INTERACTING FACTOR 4 and 5 (PIF4, PIF5), interacting with post‐translational regulation of PIF proteins by phytochrome B (phyB) and other light‐activated pathways. The model predicted bimodal and end‐of‐day PIF activity profiles that are observed across hundreds of PIF‐regulated target genes. In the response to temperature, warmth‐enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature‐dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

DNA-Binding Study Identifies C-Box and Hybrid C/G-Box or C/A-Box Motifs as High-Affinity Binding Sites for STF1 and LONG HYPOCOTYL5 Proteins

LONG HYPOCOTYL5 (HY5) is a bZIP (basic leucine zipper) transcription factor that activates photomorphogenesis and root development in Arabidopsis (Arabidopsis thaliana). Previously, STF1 (soybean [Glycine max] TGACG-motif binding factor 1), a homologous legume protein with a RING-finger motif and a bZIP domain, was reported in soybean. To investigate the role of STF1, the phenotypes of transgenic Arabidopsis plants overexpressing STF1 and HY5 were compared. In addition, the DNA-binding properties of STF1 and HY5 were extensively studied using random binding site selection and electrophoretic mobility shift assay. Overexpression of STF1 in the hy5 mutant of Arabidopsis restored wild-type photomorphogenic and root development phenotypes of short hypocotyl, accumulation of chlorophyll, and root gravitropism with partial restoration of anthocyanin accumulation. This supports that STF1 is a homolog of HY5 with a role in light and hormone signaling. The DNA-binding properties of STF1 and HY5 are shown to be similar to each other in recognizing many ACGT-containing elements with a consensus sequence motif of 5′-(G/A)(G/A) TGACGT(C/G/A)(A/T/G)-3′. The motif represents a characteristically strong preference for flanking sequence to TGACGT and a larger sequence than the sequences recognized by the G-box binding factor and TGA protein families. The finding of C-box, hybrid C/G-, and C/A-boxes as high-affinity binding sites over the G-box and parameters associated with HY5 recognition define the criteria of HY5/STF1 protein-DNA interaction in the promoter regions. This study helps to predict the precise in vivo binding sites of the HY5 protein from the vast number of putative HY5 genomic binding sites analyzed by chromatin immunoprecipitation on chip.