The evening complex is central to the difference between the circadian clocks of Arabidopsis thaliana shoots and roots

The circadian clock regulates the timing of many aspects of plant physiology, and this requires entrainment of the clock to the prevailing day:night cycle. Different plant cells and tissues can oscillate with different free‐running periods, so coordination of timing across the plant is crucial. Previous work showed that a major difference between the clock in mature shoots and roots involves light inputs. The objective of this work was to define, in Arabidopsis thaliana , the operation of the root clock in more detail, and in particular how it responds to light quality. Luciferase imaging was used to study the shoot and root clocks in several null mutants of clock components and in lines with aberrant expression of phytochromes. Mutations in each of the components of the evening complex (EARLY FLOWERING 3 and 4, and LUX ARRHYTHMO) were found to have specific effects on roots, by affecting either rhythmicity or period and its response to light quality. The data suggest that the evening complex is a key part of the light input mechanism that differs between shoots and roots and show that roots sense red light via phytochrome B

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

Manipulation of photosensory and circadian signalling restricts phenotypic plasticity in response to changing environmental conditions in Arabidopsis

Plants exploit phenotypic plasticity to adapt their growth and development to prevailing environmental conditions. Interpretation of light and temperature signals are aided by the circadian system which provides a temporal context. Phenotypic plasticity provides a selective and competitive advantage in nature but is obstructive during large-scale, intensive agricultural practices since economically important traits (including vegetative growth and flowering time) can widely vary depending on local environmental conditions. This prevents accurate prediction of harvesting times and produces a variable crop. We sought to restrict phenotypic plasticity and circadian regulation by manipulating signalling systems that govern plants’ responses to environmental signals. Mathematical modelling of plant growth and development predicted reduced plant responses to changing environments when circadian and light signaling pathways were manipulated. We tested this hypothesis by utilising a constitutively-active allele of the plant photoreceptor phytochromeB, along with disruption of the circadian system via mutation of EARLY FLOWERING3. We found that these manipulations produced plants that were less responsive to light and temperature cues and which failed to anticipate dawn. These engineered plants have uniform vegetative growth and flowering time, demonstrating how phenotypic plasticity can be limited whilst maintaining plant productivity. This has significant implications for future agriculture in both open fields and controlled environments

COLD REGULATED GENE 27 and 28 Antagonize the Transcriptional Activity of the RVE8/LNK1/LNK2 Circadian Complex

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways

PCH1 integrates circadian and light- signaling pathways to control photoperiod-responsive growth in Arabidopsis

Plants react to seasonal change in day length through altering physiology and development. Factors that function to harmonize growth with photoperiod are poorly understood. Here we characterize a new protein that associates with both circadian clock and photoreceptor components, named PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1). pch1 seedlings have overly elongated hypocotyls specifically under short days while constitutive expression of PCH1 shortens hypocotyls independent of day length. PCH1 peaks at dusk, binds phytochrome B (phyB) in a red light-dependent manner, and co-localizes with phyB into photobodies. PCH1 is necessary and sufficient to promote the biogenesis of large photobodies to maintain an active phyB pool after light exposure, potentiating red-light signaling and prolonging memory of prior illumination. Manipulating PCH1 alters PHYTOCHROME INTERACTING FACTOR 4 levels and regulates lightresponsive gene expression. Thus, PCH1 is a new factor that regulates photoperiod-responsive growth by integrating the clock with light perception pathways through modulating daily phyBsignaling

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