Identification of ASK and clock-associated proteins as molecular partners of LKP2 (LOV kelch protein 2) in Arabidopsis

The ADO/FKF/LKP/ZTL family of proteins of Arabidopsis thaliana Heynh. have a LOV domain, an F-box motif, and a kelch repeat region. LKP2 is a member of this family and functions either within or very close to the circadian oscillator in Arabidopsis. Promoter-GUS fusion studies revealed that the LKP2 gene was highly active in rosette leaves. In CaMV 35S:LKP2-GFP plants, GFP-associated fluorescence was detected in nuclei, suggesting that LKP2 is a nuclear protein. Yeast two-hybrid analysis demonstrated that LKP2 interacted with some Arabidopsis Skp1-like proteins (ASK), as do other ADO/FKF/LKP/ZTL family proteins, suggesting that LKP2 can form an SCF (Skp1-Cullin-F-box protein) complex that functions as a ubiquitin E3 ligase. LKP2 interacted not only with itself but also with other members of the family, LKP1 and FKF1. The two-hybrid analysis also demonstrated that LKP2 interacted with TOC1, a clock component, but not with CCA1 or LHY, negative regulators of TOC1 gene expression. The LOV domain of LKP2 was shown to be necessary and sufficient for the interaction with TOC1. An interaction between LKP2 and APRR5, a paralogue of TOC1, was also observed, but LKP2 did not interact with APRR3, APRR7, or APRR9, other paralogues of TOC1.     

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.     

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.     

Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis

To examine the role of the TOC1 (TIMING OF CAB EXPRESSION1) gene in the Arabidopsis circadian system, we generated a series of transgenic plants expressing a gradation in TOC1 levels. Silencing of the TOC1 gene causes arrhythmia in constant darkness and in various intensities of red light, whereas in blue light, the clock runs faster in silenced plants than in wild-type plants. Increments in TOC1 gene dosage delayed the pace of the clock, whereas TOC1 overexpression abolished rhythmicity in all light conditions tested. Our results show that TOC1 RNA interference and toc1-2 mutant plants displayed an important reduction in sensitivity to red and far-red light in the control of hypocotyl elongation, whereas increments in TOC1 gene dosage clearly enhanced light sensitivity. Furthermore, the red light-mediated induction of CCA1/LHY expression was decreased in TOC1 RNA interference and toc1-2 mutant plants, indicating a role for TOC1 in the phytochrome regulation of circadian gene expression. We conclude that TOC1 is an important component of the circadian clock in Arabidopsis with a crucial function in the integration of light signals to control circadian and morphogenic responses.     

Analysis of phase of LUCIFERASE expression reveals novel circadian quantitative trait loci in Arabidopsis

In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.     

Circadian rhythm of circumnutation in inflorescence stems of Arabidopsis

We investigate the modulation of circumnutation in inflorescence stems of Arabidopsis to determine the circadian regulation of circumnutation. Under constant light conditions (LL), circumnutation speed in wild-type plants fluctuates, with the phase of the highest speed at subjective dawn; the period length is close to 24 h. toc1 appears to shorten the period and elf3 causes an arrhythmic phenotype in circumnutation speed in LL, suggesting that a common circadian clock may control both circumnutation speed and other circadian outputs. These results highlight for the first time a role for a circadian clock in the regulation of circumnutation based on genetic analysis of Arabidopsis.     

GIGANTEA acts in blue light signaling and has biochemically separable roles in circadian clock and flowering time regulation

Circadian clocks are widespread in nature. In higher plants, they confer a selective advantage, providing information regarding not only time of day but also time of year. Forward genetic screens in Arabidopsis (Arabidopsis thaliana) have led to the identification of many clock components, but the functions of most of these genes remain obscure. To identify both new constituents of the circadian clock and new alleles of known clock-associated genes, we performed a mutant screen. Using a clock-regulated luciferase reporter, we isolated new alleles of ZEITLUPE, LATE ELONGATED HYPOCOTYL, and GIGANTEA (GI). GI has previously been reported to function in red light signaling, central clock function, and flowering time regulation. Characterization of this and other GI alleles has helped us to further define GI function in the circadian system. We found that GI acts in photomorphogenic and circadian blue light signaling pathways and is differentially required for clock function in constant red versus blue light. Gene expression and epistasis analyses show that TIMING OF CHLOROPHYLL A/B BINDING PROTEIN1 (TOC1) expression is not solely dependent upon GI and that GI expression is only indirectly affected by TOC1, suggesting that GI acts both in series with and in parallel to TOC1 within the central circadian oscillator. Finally, we found that the GI-dependent promotion of CONSTANS expression and flowering is intact in a gi mutant with altered circadian regulation. Thus GI function in the regulation of a clock output can be biochemically separated from its role within the circadian clock.     

Identification of LOV KELCH PROTEIN2 (LKP2)-interacting factors that can recruit LKP2 to nuclear bodies

LOV KELCH PROTEIN2 (LKP2) is an F-box protein that has been postulated to function centrally, or near to the circadian clock oscillator. As a first step to determine which proteins act as substrates of LKP2, yeast two-hybrid screening was performed using LKP2 as bait, and two interaction factors, Di19 and COL1, were isolated. The transiently expressed Di19-GUS fusion protein was localized in the nucleus of Arabidopsis petiole cells. COL1 and other CO/COL family proteins could also interact with LKP1/ZTL, LKP2 or FKF1. The LKP2-binding site in CO or COL1 was near the center of each protein. The CCT motif in CO or COL1 was not sufficient for interaction with LKP2. LKP2 recognized CO with F-box and kelch repeat-containing regions, while it recognized COL1 with an LOV domain. When LKP2 was fused with cyan fluorescent proein (CFP) and transiently expressed in onion epidermal cells, CFP-LKP2 signals were localized in the nucleus and cytosol. Both yellow fluorescent protein (YFP)-CO and YFP-COL1 were located in the nucleus, forming nuclear bodies when they were transiently expressed. However, co-expression of CFP-LKP2 with YFP fused to either CO or COL1 resulted in the recruitment of CFP-LKP2 in nuclear bodies. Furthermore, the CFP-LKP2 and YFP-CO signals co-localized with signals for pU2B'-mRFP, which is a marker for Cajal bodies. These results suggest the possibility that LKP2 functions with CO/COL family proteins in the nuclear bodies.     

Circadian Clock-Regulated Expression of Phytochrome and Cryptochrome Genes in Arabidopsis

Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 h. Endogenous oscillators called circadian clocks regulate these rhythms. The circadian clocks are synchronized to the periodic environmental changes (e.g. day/night cycles) by specific stimuli; among these, the most important is the light. Photoreceptors, phytochromes, and cryptochromes are involved in setting the clock by transducing the light signal to the central oscillator. In this work, we analyzed the spatial, temporal, and long-term light-regulated expression patterns of the Arabidopsis phytochrome (PHYA to PHYE) and cryptochrome (CRY1 and CRY2) promoters fused to the luciferase (LUC(+)) reporter gene. The results revealed new details of the tissue-specific expression and light regulation of the PHYC and CRY1 and 2 promoters. More importantly, the data obtained demonstrate that the activities of the promoter::LUC(+) constructs, with the exception of PHYC::LUC(+), display circadian oscillations under constant conditions. In addition, it is shown by measuring the mRNA abundance of PHY and CRY genes under constant light conditions that the circadian control is also maintained at the level of mRNA accumulation. These observations indicate that the plant circadian clock controls the expression of these photoreceptors, revealing the formation of a new regulatory loop that could modulate gating and resetting of the circadian clock.     

XAP5 CIRCADIAN TIMEKEEPER coordinates light signals for proper timing of photomorphogenesis and the circadian clock in Arabidopsis

Numerous, varied, and widespread taxa have an internal circadian clock that allows anticipation of rhythmic changes in the environment. We have identified XAP5 CIRCADIAN TIMEKEEPER (XCT), an Arabidopsis thaliana gene important for light regulation of the circadian clock and photomorphogenesis. XCT is essential for proper clock function: xct mutants display a shortened circadian period in all conditions tested. Interestingly, XCT plays opposite roles in plant responses to light depending both on trait and wavelength. The clock in xct plants is hypersensitive to red but shows normal responses to blue light. By contrast, inhibition of hypocotyl elongation in xct is hyposensitive to red light but hypersensitive to blue light. Finally, XCT is important for ribulose-1,5-bisphosphate carboxylase/oxygenase production and plant greening in response to light. This novel combination of phenotypes suggests XCT may play a global role in coordinating growth in response to the light environment. XCT contains a XAP5 domain and is well conserved across diverse taxa, suggesting it has a common function in higher eukaryotes. Downregulation of the XCT ortholog in Caenorhabditis elegans is lethal, suggesting that studies in Arabidopsis may be instrumental to understanding the biochemical activity of XCT.     

GIGANTEA and SPINDLY genes linked to the clock pathway that controls circadian characteristics of transpiration in Arabidopsis

Several "clock" genes that regulate the circadian system in Arabidopsis thaliana have been identified. The GIGANTEA (GI) gene has been shown to participate in the circadian system that is linked to overt rhythms in gene expression, leaf movements, hypocotyl elongation, and photoperiodic control of flowering in Arabidopsis. During continuous light (LL), circadian expression patterns in gi-2 mutants show reduced amplitudes and altered period lengths when compared with controls. Rhythms in stomatal function, such as transpiration, have been shown to be endogenous and persist in constant lighting conditions. In order to test for a physiologic variable that might be affected by the circadian clock via the GI gene, we compared circadian characteristics of transpiration between three Arabidopsis mutants (gi-2, spy-4, spy-4/gi-2) and wild-type (WT) controls in synchronized (LD for 2.5d) and free-running (LL for 3d) conditions. Each genotype showed a significant circadian rhythm in LD at p < 0.001, with acrophases located near the middle of the daily 14h L-span, with average amplitudes for WT: 18.9%, gi-2: 16.1%, spy-4: 7.7%, and spy-4/gi-2: 5.3%. On the first day in LL, the circadian amplitude was dramatically reduced to 3.1% for gi-2 compared with WT (11.9%), while amplitudes for spy-4 (6.9%) and spy-4/gi-2 (5.7%) were not significantly changed from LD. The amplitude for gi-2 remained low during days 2 (4.2%) and 3 (2.1%) in LL, while it slowly dampened for the WT (8.6 and 6.6%). The amplitudes for spy-4 (6.6%) and spy-4/gi-2 (5.6%) on day 2 in LL were indistinguishable from the LD span, but finally dampened on day 3 in LL (1.9 and 2.3%, respectively). These data suggest that transpiration is a physiologic variable controlled by a circadian system that involves both the GI and SPY proteins.     

Circadian timekeeping during early Arabidopsis development

The circadian coordination of organismal biology with the local temporal environment has consequences for fitness that may become manifest early in development. We directly explored the development of the Arabidopsis (Arabidopsis thaliana) clock in germinating seedlings by monitoring expression of clock genes. Clock function is detected within 2 d of imbibition (hydration of the dried seed). Imbibition is sufficient to synchronize individuals in a population in the absence of entraining cycles of light-dark or temperature, although light-dark and temperature cycles accelerate the appearance of rhythmicity and improve synchrony among individuals. Oscillations seen during the first 2 d following imbibition are dependent on the clock genes LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, ZEITLUPE, GIGANTEA, PSEUDO-RESPONSE REGULATOR7 (PRR7), and PRR9, although later circadian oscillations develop in mutants defective in each of these genes. In contrast to circadian rhythmicity, which developed under all conditions, amplitude was the only circadian parameter that demonstrated a clear response to the light environment; clock amplitude is low in the dark and high in the light. A circadian clock entrainable by temperature cycles in germinating etiolated seedlings may synchronize the buried seedling with the local daily cycles before emergence from the soil and exposure to light.     

Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock

To identify new components in the phytochrome (phy) signaling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were isolated that exhibited reduced sensitivity to both continuous red and far-red light, suggesting involvement in both phyA and phyB signaling. The molecular lesions responsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) gene. PRR7 is a member of a small gene family in Arabidopsis previously suggested to be involved in circadian rhythms. A PRR7-beta-glucuronidase fusion protein localized to the nucleus, implying a possible function in the regulation of photoresponsive gene expression. Consistent with this suggestion, prr7 seedlings were partially defective in the regulation of the rapidly light-induced genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), observable as a premature increase in expression level during the second peak of the biphasic induction profile that is elicited upon initial exposure of dark-grown seedlings to light. A similar 3- to 6-h coordinated advance in peak free-running expression of CCA1, LHY, and TIMING-OF-CAB1, which are considered to encode the molecular components of the circadian oscillator in Arabidopsis, was observed in entrained fully green prr7 seedlings compared with wild-type seedlings. Collectively, these data suggest that PRR7 functions as a signaling intermediate in the phytochrome-regulated gene expression responsible for both seedling deetiolation and phasing of the circadian clock in response to light.     

Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.