Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways

Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis

Protein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperature

A mobile ELF4 delivers circadian temperature information from shoots to roots

Extended Data and Source Data can be found at https://doi.org/10.1038/s41477-020-0634-2Ajuts: the Mas laboratory is funded by the FEDER/Spanish Ministry of Economy and Competitiveness, the Ramon Areces Foundation and the Generalitat de Catalunya (AGAUR). The P.M. laboratory also acknowledges financial support from the CERCA Program, Generalitat de Catalunya and by the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa Program for Centers of Excellence in R&D 2016-2019 (SEV-2015-0533).The circadian clock is synchronized by environmental cues, mostly by light and temperature. Explaining how the plant circadian clock responds to temperature oscillations is crucial to understanding plant responsiveness to the environment. Here, we found a prevalent temperature-dependent function of the Arabidopsis clock component EARLY FLOWERING 4 (ELF4) in the root clock. Although the clocks in roots are 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 for controlling the period of the root clock in a temperature-dependent manner. Low temperatures favour ELF4 mobility, resulting in a slow-paced root clock, whereas 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 root

Chromatin dynamics and transcriptional control of circadian rhythms in arabidopsis

Circadian rhythms pervade nearly all aspects of plant growth, physiology, and development. Generation of the rhythms relies on an endogenous timing system or circadian clock that generates 24-hour oscillations in multiple rhythmic outputs. At its bases, the plant circadian function relies on dynamic interactive networks of clock components that regulate each other to generate rhythms at specific phases during the day and night. From the initial discovery more than 13 years ago of a parallelism between the oscillations in chromatin status and the transcriptional rhythms of an Arabidopsis clock gene, a number of studies have later expanded considerably our view on the circadian epigenome and transcriptome landscapes. Here, we describe the most recent identification of chromatin-related factors that are able to directly interact with Arabidopsis clock proteins to shape the transcriptional waveforms of circadian gene expression and clock outputs. We discuss how changes in chromatin marks associate with transcript initiation, elongation, and the rhythms of nascent RNAs, and speculate on future interesting research directions in the field

Molecular mechanism of photoperiod sensing

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well-studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth during the night and the day, respectively. We also provide evidence that ELF3, for which so far only a growth inhibitory role has been reported, can also act as a growth promoter under certain conditions. Finally, circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development is completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by manipulating the combination of these two key genes

A molecular basis of ELF3 action in the Arabidopsis circadian clock

The circadian clock anticipates daily environmental changes and optimizes timing of physiological events. Circadian systems are present in most living organisms. In Arabidopsis, circadian components are arranged in positive/negative regulatory feed-back loops. The core loop is arranged by the morning transcription factors LHY and CCA1, and the evening pseudo-response regulator TOC1. The morning loop reciprocally connects LHY and CCA1 to the TOC1-sequence-related components PRR9 and PRR7. Other genes, when mutated, display a clock phenotype, but not all of such genes have been placed into the clock model. For instance, three evening genes, ELF3, ELF4, and LUX, have been found to be essential for circadian function. Clock- and light-signaling networks are tightly interconnected. Light input to the clock is mediated by photoreceptors, such as the phytochromes. ELF3 and ELF4 play a pivotal role in the generation of circadian rhythms and in the integration of light signal to the clock mechanism. Both encoded proteins are reported to be located in the nucleus, and both the elf3 and the elf4 mutants display a similarly arrested clock. In this thesis, ELF3 was found to be genetically downstream of ELF4 within the same clock signaling pathway required to sustain circadian rhythms. Moreover, I found that ELF3 and ELF4 proteins physically interact. This interaction correlated with an increase of ELF3 nuclear localization. These observations are consistent with a role of ELF4 as an effector that promotes ELF3 activity to lengthen circadian periodicity. A functional complementation approach identified three functional modules in the ELF3 encoded protein. The N-terminus and middle domains mediate interaction with phyB and ELF4, respectively. The C-terminus domain was found to be required for ELF3 nuclear localization. Thus, ELF3 is a multifunctional protein that interacts with both light-signaling and clock components. The molecular function of ELF3 had previously remained elusive. PRR9 expression was found to be down-regulated in ELF3 and ELF4 over-expressors. Interestingly, I found that ELF3 physically associated with the same conserved region in the PRR9 promoter as the transcription factor LUX. I found that LUX was genetically downstream of ELF4, and that LUX required ELF3. Taken together, I proposed that ELF3, ELF4, and LUX are part of an evening-clock complex required to repress PRR9 expression, and to sustain circadian oscillations. ELF3 has been reported to be crucial to buffer light input to the oscillator. Photoreceptors and ELF3 play an opposite role in light-mediated acceleration of circadian periodicity, where photoreceptors shortens, and ELF3 lengthens, circadian period under constant light. Interestingly, I found that the N-terminus of ELF3 was not essential for ELF3 circadian function, but that mediated the physical interaction of ELF3 to phyB. An elf3 complementation line deleted for its N-terminus displayed hyposensitivity to the period-shortening effect induced by constant-red light. Therefore, I hypothesized that phyB interaction to the N-terminus of ELF3 mediates light-repression of ELF3 action in circadian-periodicity. In chapter 4, further characterization of the weak allele elf3-12 supported the role of ELF3 as a decelerator of circadian periodicity. The elf3-12 mutation encodes an amino-acid replacement in a conserved box within the ELF4-binding domain. The elf3 12 coding region led to robust expression of ELF3-12 protein, and ELF3-12 retained the capacity to bind both ELF4 and phyB. elf3-12 displayed light-dependent short-period phenotype that was enhanced by phytochrome over-expression. Moreover, elf3-12 displayed hypersensitive to red-light-resetting pulses. Thus, I found that elf3-12 is attenuated in its function to repress light input to the clock and/or and increased phy-mediated repression of ELF3 function. elf3-12 was the first described elf3 weak allele. My characterization of a collection of elf3 TILLING alleles led to the identification of novel short- and long-period alleles that I predict will expand current understanding of the role of ELF3 as an integrator of light signals and as a core-clock component. Taken together, my thesis has placed ELF3 within the circadian mechanism. ELF3, ELF4, and LUX are part of an evening-repressor complex required to sustain circadian function. The genetic interaction of these three genes is consistent with a hierarchy of complex assembly. In this, I propose that ELF4 works as an effector protein that activates ELF3, possibly by increasing the ELF3 nuclear pool. Then, the association of both ELF3 and LUX to the PRR9 promoter is required for transcriptional repression of PRR9. Additionally, I propose that ELF3 function in circadian periodicity is modulated by its interaction partners by a competition between a positive effect of ELF4 and a light-mediated-negative effect of phyB. This is consistent with ELF3 being a multifunctional protein that integrates light signals as a core-oscillator component

Molecular Mechanisms Underlying the Arabidopsis Circadian Clock

A wide range of biological processes exhibit circadian rhythm, enabling plants to adapt to the environmental day–night cycle. This rhythm is generated by the so-called ‘circadian clock’. Although a number of genetic approaches have identified >25 clock-associated genes involved in the Arabidopsis clock mechanism, the molecular functions of a large part of these genes are not known. Recent comprehensive studies have revealed the molecular functions of several key clock-associated proteins. This progress has provided mechanistic insights into how key clock-associated proteins are integrated, and may help in understanding the essence of the clock's molecular mechanisms

Circadian clock signaling in Arabidopsis thaliana : From gene expression to physiology and development

The daily rotation of the earth on its axis leads to predictable periodic fluctuations of environmental conditions. Accordingly, most organisms have evolved an internal timing mechanism, the circadian clock, which is able to recognize these 24-hour rhythmic oscillations. In plants, the temporal synchronization of physiology with the environment is essential for successful plant growth and development. The intimate connection between light signaling pathways and the circadian oscillator allows the anticipation of the environmental transitions and the measurement of day-length as an indicator of changing seasons. In recent years, significant advances have been made in the genetic and molecular dissection of the plant circadian system, mostly in Arabidopsis thaliana. The overall plant clock organization is highly complex; the system seems to include several input pathways, tightly regulated central oscillators and a myriad of outputs. The molecular cloning and characterization of a number of clock components has greatly improved our view of the plant central oscillator and additional players will most likely come into place very soon. Molecular mechanisms underlying circadian clock function are also beginning to be characterized. The emerging model relies on negative feedback loops at the core of the oscillator. Additional levels of post-transcriptional and post-translational regulation also contribute to the generation and maintenance of the rhythms. Globally, these studies have shed new light on how the clock coordinates plant physiology and development with the daily and seasonal environmental cycles

Accurate timekeeping is controlled by a cycling activator in Arabidopsis.

Transcriptional feedback loops are key to circadian clock function in many organisms. Current models of the Arabidopsis circadian network consist of several coupled feedback loops composed almost exclusively of transcriptional repressors. Indeed, a central regulatory mechanism is the repression of evening-phased clock genes via the binding of morning-phased Myb-like repressors to evening element (EE) promoter motifs. We now demonstrate that a related Myb-like protein, REVEILLE8 (RVE8), is a direct transcriptional activator of EE-containing clock and output genes. Loss of RVE8 and its close homologs causes a delay and reduction in levels of evening-phased clock gene transcripts and significant lengthening of clock pace. Our data suggest a substantially revised model of the circadian oscillator, with a clock-regulated activator essential both for clock progression and control of clock outputs. Further, our work suggests that the plant clock consists of a highly interconnected, complex regulatory network rather than of coupled morning and evening feedback loops. DOI:http://dx.doi.org/10.7554/eLife.00473.001

Plant Defense against Insect Herbivory is Mediated by the Circadian Clock

Organisms on earth evolved a circadian clock that matches the planet's 24-hour rotation. The plant clock controls many behaviors and proper entrainment of the clock to the environment leads to a competitive overall growth advantage. Despite the finding that many wound-inducible genes are also circadian regulated, it was uncertain whether this regulation is important for plant defense against herbivorous insects. We found that plants entrained to light-dark cycles 12 hours out of phase with the predator, Trichoplusia ni (cabbage loopers), were more susceptible to T. ni herbivory than plants entrained in phase with T ni . In contrast, arrhythmic clock and jasmonate-deficient mutants were equally susceptible to T. ni herbivory whether entrained in the same or reciprocal 12-hour light-dark cycles. These results suggest that the circadian rhythms, acting through jasmonate signals and the clock, add selective advantage to plants through enhanced anticipation of and defense against herbivory