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 regulates light, temperature, and circadian signaling as a structural component of phytochrome B-photobodies in Arabidopsis.

The members of the phytochrome (phy) family of bilin-containing photoreceptors are major regulators of plant photomorphogenesis through their unique ability to photointerconvert between a biologically inactive red light-absorbing Pr state and an active far-red light-absorbing Pfr state. While the initial steps in Pfr signaling are unclear, an early event for the phyB isoform after photoconversion is its redistribution from the cytoplasm into subnuclear foci known as photobodies (PBs), which dissipate after Pfr reverts back to Pr by far-red irradiation or by temperature-dependent nonphotochemical reversion. Here we present evidence that PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) functions both as an essential structural component of phyB-containing PBs and as a direct regulator of thermal reversion that is sufficient to stabilize phyB as Pfr in vitro. By examining the genetic interaction between a constitutively active phyBY276H-YFP allele (YHB-YFP) and PCH1, we show that the loss of PCH1 prevents YHB from coalescing into PBs without affecting its nuclear localization, whereas overexpression of PCH1 dramatically increases PB levels. Loss of PCH1, presumably by impacting phyB-PB assembly, compromises a number of events elicited in YHB-YFP plants, including their constitutive photomorphogenic phenotype, red light-regulated thermomorphogenesis, and input of phyB into the circadian clock. Conversely, elevated levels of both phyB and PCH1 generate stable, yet far-red light-reversible PBs that persisted for days. Collectively, our data demonstrate that the assembly of PCH1-containing PBs is critical for phyB signaling to multiple outputs and suggest that altering PB dynamics could be exploited to modulate plant responses to light and temperature.

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.

Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots.

The OLIGOPEPTIDE TRANSPORTER 3 (OPT3) has recently been identified as a component of the systemic network mediating iron (Fe) deficiency responses in Arabidopsis. Reduced expression of OPT3 induces an over accumulation of Fe in roots and leaves, due in part by an elevated expression of the IRON-REGULATED TRANSPORTER 1. Here we show however, that opt3 leaves display a transcriptional program consistent with an Fe overload, suggesting that Fe excess is properly sensed in opt3 leaves and that the OPT3-mediated shoot-to-root signaling is critical to prevent a systemic Fe overload. We also took advantage of the tissue-specific localization of OPT3, together with other Fe-responsive genes, to determine the timing and location of early transcriptional events during Fe limitation and resupply. Our results show that the leaf vasculature responds more rapidly than roots to both Fe deprivation and resupply, suggesting that the leaf vasculature is within the first tissues that sense and respond to changes in Fe availability. Our data highlight the importance of the leaf vasculature in Fe homeostasis by sensing changes in apoplastic levels of Fe coming through the xylem and relaying this information back to roots via the phloem to regulate Fe uptake at the root level.

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.

Cross-species complementation reveals conserved functions for EARLY FLOWERING 3 between monocots and dicots.

Plant responses to the environment are shaped by external stimuli and internal signaling pathways. In both the model plant Arabidopsis thaliana (Arabidopsis) and crop species, circadian clock factors are critical for growth, flowering, and circadian rhythms. Outside of Arabidopsis, however, little is known about the molecular function of clock gene products. Therefore, we sought to compare the function of Brachypodium distachyon (Brachypodium) and Setaria viridis (Setaria) orthologs of EARLY FLOWERING 3, a key clock gene in Arabidopsis. To identify both cycling genes and putative ELF3 functional orthologs in Setaria, a circadian RNA-seq dataset and online query tool (Diel Explorer) were generated to explore expression profiles of Setaria genes under circadian conditions. The function of ELF3 orthologs from Arabidopsis, Brachypodium, and Setaria was tested for complementation of an elf3 mutation in Arabidopsis. We find that both monocot orthologs were capable of rescuing hypocotyl elongation, flowering time, and arrhythmic clock phenotypes. Using affinity purification and mass spectrometry, our data indicate that BdELF3 and SvELF3 could be integrated into similar complexes in vivo as AtELF3. Thus, we find that, despite 180 million years of separation, BdELF3 and SvELF3 can functionally complement loss of ELF3 at the molecular and physiological level.

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 light-responsive gene expression. Thus, PCH1 is a new factor that regulates photoperiod-responsive growth by integrating the clock with light perception pathways through modulating daily phyB-signaling.