Complex epistatic interactions between ELF3, PRR9, and PRR7 regulate the circadian clock and plant physiology.

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7.

LNKs-RVEs complex ticks in the circadian gating of plant temperature stress responses.

Recently, Kidokoro et al. found that protein complex LNK3,4-RVE4,8 and LNK1,2-RVE4,8 of the circadian clock modulates plant cold- and high-temperature tolerance, respectively. Here, we reviewed the discovery of LNKs, the dynamically formed morning-phased clock complexes, and their critical role on endogenous circadian rhythms.

The circadian clock ticks in plant stress responses.

The circadian clock, a time-keeping mechanism, drives nearly 24-h self-sustaining rhythms at the physiological, cellular, and molecular levels, keeping them synchronized with the cyclic changes of environmental signals. The plant clock is sensitive to external and internal stress signals that act as timing cues to influence the circadian rhythms through input pathways of the circadian clock system. In order to cope with environmental stresses, many core oscillators are involved in defense while maintaining daily growth in various ways.

Circadian clock in plants: Linking timing to fitness.

Endogenous circadian clock integrates cyclic signals of environment and daily and seasonal behaviors of organisms to achieve spatiotemporal synchronization, which greatly improves genetic diversity and fitness of species. This review addresses recent studies on the plant circadian system in the field of chronobiology, covering topics on molecular mechanisms, internal and external Zeitgebers, and hierarchical regulation of physiological outputs. The architecture of the circadian clock involves the autoregulatory transcriptional feedback loops, post-translational modifications of core oscillators, and epigenetic modifications of DNA and histones.

Firefly Luciferase Complementation-Based Analysis of Dynamic Protein-Protein Interactions Under Diurnal and Circadian Conditions in Arabidopsis.

Split firefly luciferase complementation assay (FLCA) is one of the most widely used sensitive and reliable methods for the analysis of constitutive and dynamic protein-protein interactions (PPIs). Here, we report a method for long-term in vivo detects plant protein-protein interactions in Arabidopsis F1 hybrids via Topcount™ Microplate Scintillation Counter or Deep-Cooled CCD camera. Following these protocols, we successfully detected time-dependent PPIs of EARLY FLOWERING 3 (ELF3) and EARLY FLOWERING 4 (ELF4); both of them with LUX ARRHYTHMO (LUX) belong to an evening complex which has been found to play a key role in circadian rhythms, flowering, and growth.

Measurement of Luciferase Rhythms in Soybean Hairy Roots.

Firefly luciferase is widely used as a bioluminescence reporter, which is simple, high signal-to-noise ratio and especially suitable for the long-term analysis of circadian clock-regulated gene expression. Here, we report the method of tracking circadian rhythms in Agrobacterium rhizogenes-induced soybean hairy roots via TopCount™ Microplate Scintillation Counter or Deep-Cooled CCD camera. Using transgenic soybean hairy roots, we monitored the endogenous 24-h oscillations of clock genes expression and investigated the precise parameters of circadian rhythmicity.

Circadian Rhythm: Phase Response Curve and Light Entrainment.

The circadian clock responds to light signals and therefore participates in the plant's daily response to light. The phase response curve (PRC) is typically used in the study of chronobiology to detect the effect of various environmental cues on a given circadian rhythm. In this chapter we describe protocols on measuring the setting of the light pulses at different times of a day, the measurement of circadian rhythm, and the calculation of phase shift in response to light pulses.

BBX19 fine-tunes the circadian rhythm by interacting with PSEUDO-RESPONSE REGULATOR proteins to facilitate their repressive effect on morning-phased clock genes.

The core plant circadian oscillator is composed of multiple interlocked transcriptional-translational feedback loops, which synchronize endogenous diel physiological rhythms to the cyclic changes of environmental cues. PSEUDO-RESPONSE REGULATORS (PRRs) have been identified as negative components in the circadian clock, though their underlying molecular mechanisms remain largely unknown. Here, we found that a subfamily of zinc finger transcription factors, B-box (BBX)-containing proteins, have a critical role in fine-tuning circadian rhythm.

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation.

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the locus, which confer extended flowering phase and thereby improve yield. The identity of as an ortholog of , a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles.

Recognition of CCA1 alternative protein isoforms during temperature acclimation.

CCA1α and CCA1β protein variants respond to environmental light and temperature cues, and higher temperature promotes CCA1β protein production and causes its retention detectable in the cytoplasm. CIRCADIAN CLOCK ASSOCIATED1 (CCA1), as the core transcription factor of circadian clock, is involved in the regulation of endogenous circadian rhythm in Arabidopsis. Previous studies have shown that CCA1 consists of two abundant splice variants, fully spliced CCA1α and intron-retaining CCA1β.

and negatively regulate the expression of EC components under warm temperature in roots.

Circadian clock operates autonomously in each cell and drives the approximately 24-h rhythm in individual tissues and organs. It is known that the evening complex (EC) components and GI are required for ambient temperature perception and thermomorphogenesis in higher plants. Our previous study found that and are required for the lengthened period of the circadian rhythm in roots, and they are responsible for the temperature overcompensation in shoots.

Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean domestication.

Adaptive changes in plant phenology are often considered to be a feature of the so-called 'domestication syndrome' that distinguishes modern crops from their wild progenitors, but little detailed evidence supports this idea. In soybean, a major legume crop, flowering time variation is well characterized within domesticated germplasm and is critical for modern production, but its importance during domestication is unclear. Here, we identify sequential contributions of two homeologous pseudo-response-regulator genes, Tof12 and Tof11, to ancient flowering time adaptation, and demonstrate that they act via LHY homologs to promote expression of the legume-specific E1 gene and delay flowering under long photoperiods.

Molecular investigation of organ-autonomous expression of Arabidopsis circadian oscillators.

The circadian pacemaker in plants is a hierarchical multioscillator system that directs and maintains a 24-hr oscillation required for organism homeostasis and environmental fitness. Molecular clockwork within individual tissues and organs acts cell autonomously, showing differences in circadian expression of core oscillators and their target genes; there are functional dominance and coupling in the complex regulatory network. However, molecular characteristics of organ-specific clocks are still unknown.

Light- and temperature-entrainable circadian clock in soybean development.

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli.

COR27 and COR28 encode nighttime repressors integrating Arabidopsis circadian clock and cold response.

It was noted that circadian components function in plant adaptation to diurnal temperature cycles and freezing tolerance. Our genome-wide transcriptome analysis revealed that evening-phased COR27 and COR28 mainly repress the transcription of clock-associated evening genes PRR5, ELF4 and cold-responsive genes. Chromatin immunoprecipitation indicated that CCA1 is recruited to the site containing EE elements of COR27 and COR28 promoters in a temperature-dependent way.

Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

"pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro.

LNK1 and LNK2 recruitment to the evening element require morning expressed circadian related MYB-like transcription factors.

Transcriptional feedback loops in Arabidopsis circadian clock is composed of more repressive components, while the knowledge of activation mechanism remains limited. We recently reported 2 members from a family of NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED genes, LNK1 and LNK2, dynamically interact with morning-phased transcriptional factors, like CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), REVEILLE8 (RVE8) and RVE4, and function as coactivators for the expression of TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5) via transcriptional factors RVE8 and RVE4. Here we provide evidence that both LNK1 and LNK2 play critical role in the transcriptional activation of PRR5, LNK1 may contribute more than LNK2 did under experimental conditions.

Allelic polymorphism of GIGANTEA is responsible for naturally occurring variation in circadian period in Brassica rapa.

GIGANTEA (GI) was originally identified by a late-flowering mutant in Arabidopsis, but subsequently has been shown to act in circadian period determination, light inhibition of hypocotyl elongation, and responses to multiple abiotic stresses, including tolerance to high salt and cold (freezing) temperature. Genetic mapping and analysis of families of heterogeneous inbred lines showed that natural variation in GI is responsible for a major quantitative trait locus in circadian period in Brassica rapa. We confirmed this conclusion by transgenic rescue of an Arabidopsis gi-201 loss of function mutant.

LNK1 and LNK2 are transcriptional coactivators in the Arabidopsis circadian oscillator.

Transcriptional feedback loops are central to the architecture of eukaryotic circadian clocks. Models of the Arabidopsis thaliana circadian clock have emphasized transcriptional repressors, but recently, Myb-like REVEILLE (RVE) transcription factors have been established as transcriptional activators of central clock components, including PSEUDO-RESPONSE REGULATOR5 (PRR5) and TIMING OF CAB EXPRESSION1 (TOC1). We show here that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2, members of a small family of four LNK proteins, dynamically interact with morning-expressed oscillator components, including RVE4 and RVE8.

A Role for CHH Methylation in the Parent-of-Origin Effect on Altered Circadian Rhythms and Biomass Heterosis in Arabidopsis Intraspecific Hybrids.

Hybrid plants and animals often show increased levels of growth and fitness, a phenomenon known as hybrid vigor or heterosis. Circadian rhythms optimize physiology and metabolism in plants and animals. In plant hybrids and polyploids, expression changes of the genes within the circadian regulatory network, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), lead to heterosis.

Measurement of luciferase rhythms.

Firefly luciferase (LUC) is a sensitive and versatile reporter for the analysis of gene expression. Transgenic plants carrying CLOCK GENE promoter:LUC fusions can be assayed with high temporal resolution. LUC measurement is sensitive, noninvasive, and nondestructive and can be readily automated, greatly facilitating genetic studies.

Variation in Arabidopsis flowering time associated with cis-regulatory variation in CONSTANS.

The onset of flowering, the change from vegetative to reproductive development, is a major life history transition in flowering plants. Recent work suggests that mutations in cis-regulatory mutations should play critical roles in the evolution of this (as well as other) important adaptive traits, but thus far there has been little evidence that directly links regulatory mutations to evolutionary change at the species level. While several genes have previously been shown to affect natural variation in flowering time in Arabidopsis thaliana, most either show protein-coding changes and/or are found at low frequency (<5%).

Crosstalk between the circadian clock and innate immunity in Arabidopsis.

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity.

SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis.

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function.