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. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we first employed mathematical modeling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrhythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modeling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod-insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship among plant circadian genes.

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. In addition, we summarized the research work on LNKs with the interacting proteins RVEs, CCA1 in temperature responses and discussed how the circadian clock confer increased fitness via gating the rhythmic expression of their target genes.

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. Here, light, temperature, humidity, and internal elemental nutrients are summarized to illustrate the sensitivity of the circadian clock to timing cues. In addition, the circadian clock runs cell-autonomously, driving independent circadian rhythms in various tissues. The core oscillators responds to each other with biochemical factors including calcium ions, mineral nutrients, photosynthetic products, and hormones. We describe clock components sequentially expressed during a 24-h day that regulate rhythmic growth, aging, immune response, and resistance to biotic and abiotic stresses. Notably, more data have suggested the circadian clock links chrono-culture to key agronomic traits in crops.

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. Recent studies have shown that a hierarchical multi-oscillator network orchestrates the defense through rhythmic accumulation of gene transcripts, alternative splicing of mRNA precursors, modification and turnover of proteins, subcellular localization, stimuli-induced phase separation, and long-distance transport of proteins. This review summarizes the essential role of circadian core oscillators in response to stresses in Arabidopsis thaliana and crops, including daily and seasonal abiotic stresses (low or high temperature, drought, high salinity, and nutrition deficiency) and biotic stresses (pathogens and herbivorous insects). By integrating time-keeping mechanisms, circadian rhythms and stress resistance, we provide a temporal perspective for scientists to better understand plant environmental adaptation and breed high-quality crop germplasm for agricultural production.

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. The promoter:luciferase reporter was used to provide fine rhythmic traces and the subsequent circadian parameters of mathematical analysis. A classical PRC assay to light pulses is the key experimental basis for determining the signal components of resetting the circadian clock.

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. Researchers can easily analyze the circadian phenotype in legumes and non-legumes using bioluminescence reporters carried by the hairy roots, avoiding time-consuming transgenic work.

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.

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. We demonstrated that overexpressing Arabidopsis thaliana BBX19 and BBX18 significantly lengthened the circadian period, while the null mutation of BBX19 accelerated the circadian speed. Moreover, BBX19 and BBX18, which are expressed during the day, physically interacted with PRR9, PRR7, and PRR5 in the nucleus in precise temporal ordering from dawn to dusk, consistent with the respective protein accumulation pattern of PRRs. Our transcriptomic and genetic analysis indicated that BBX19 and PRR9, PRR7, and PRR5 cooperatively inhibited the expression of morning-phased clock genes. PRR proteins affected BBX19 recruitment to the CCA1, LHY, and RVE8 promoters. Collectively, our findings show that BBX19 interacts with PRRs to orchestrate circadian rhythms, and suggest the indispensable role of transcriptional regulators in fine-tuning the circadian clock.

Multi-omic dissection of the drought resistance traits of soybean landrace LX.

With diverse genetic backgrounds, soybean landraces are valuable resource for breeding programs. Herein, we apply multi-omic approaches to extensively characterize the molecular basis of drought tolerance in the soybean landrace LX. Initial screens established that LX performed better with PEG6000 treatment than control cultivars. LX germinated better than William 82 under drought conditions and accumulated more anthocyanin and flavonoids. Untargeted mass spectrometry in combination with transcriptomic analyses revealed the chemical diversity and genetic basis underlying the overall performance of LX landrace. Under control and drought conditions, significant differences in the expression of a suite of secondary metabolism genes, particularly those involved in the general phenylpropanoid pathway and flavonoid but not lignin biosynthesis, were seen in LX and William 82. The expression of these genes correlated with the corresponding metabolites in LX plants. Further correlation analysis between metabolites and transcripts identified pathway structural genes and transcription factors likely are responsible for the LX agronomic traits. The activities of some key biosynthetic genes or regulators were confirmed through heterologous expression in transgenic Arabidopsis and hairy root transformation in soybean. We propose a regulatory mechanism based on flavonoid secondary metabolism and adaptive traits of this landrace which could be of relevance to cultivated soybean.

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 J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

Recognition of CCA1 alternative protein isoforms during temperature acclimation.

KEY MESSAGE: CCA1α and CCA1ß protein variants respond to environmental light and temperature cues, and higher temperature promotes CCA1ß 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 CCA1ß. CCA1ß is believed to form a nonfunctional heterodimer with CCA1α and its closed-related homolog LHY. Many studies have established that CCA1ß is a transcription product, while how CCA1ß protein is produced and how two CCA1 isoforms respond to environmental cues have not been elucidated. In this study, we identified CCA1α and CCA1ß protein variants under different photoperiods with warm or cold temperature cycles, respectively. Our results showed that CCA1 protein production is regulated by prolonged light exposure and warm temperature. The protein levels of CCA1α and CCA1ß peak in the morning, but the detection of CCA1ß is dependent on immunoprecipitation enrichment at 22 °C. Higher temperature of 37 °C promotes CCA1ß protein production and causes its retention to be detectable in the cytoplasm. Overall, our results indicate that two splice variants of the CCA1 protein respond to environmental light and temperature signals and may, therefore, maintain the circadian rhythms and give individuals the ability to adapt to environment.

PRR9 and PRR7 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 PRR9 and 7 are required for the lengthened period of the circadian rhythm in roots, and they are responsible for the temperature overcompensation in shoots. However, the molecular mechanism of the circadian clock, especially in different tissues, in response to temperature oscillations remains largely unknown. Here, we studied the transcript levels of EC genes and GI of the prr7 prr9 mutant shoots and roots in response to 22°C or 28°C, respectively. The results showed that PRR9, 7 in roots inhibited the transcripts accumulation of ELF3, ELF4, and LUX at 28°C. In addition, loss-of-function of both PRR9 and 7 caused an increase in GI expression at 22°C, but warm temperature of 28°C limited the negative effect of PRR9, 7 on GI in roots. Our findings proposed a temperature-dependent molecular basis for root-specific circadian clock and indicated the critical role for PRR9, 7 in negatively regulating ELF3, ELF4, LUX, and GI in the circadian gating of thermoresponse.

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. We show that Tof12-dependent acceleration of maturity accompanied a reduction in dormancy and seed dispersal during soybean domestication, possibly predisposing the incipient crop to latitudinal expansion. Better understanding of this early phase of crop evolution will help to identify functional variation lost during domestication and exploit its potential for future crop improvement.

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. Here, we showed the detached shoot and root possess dynamic circadian protein-protein interactions between clock core components, periodicity in organs exhibits a difference. The period length difference between shoot and root was not remarkable in prr7-3 and prr7-3 prr9-1 mutants. In addition, the phase transition curve indicated that shoot and root clock respond differently to the resetting cues of ambient temperature. PRR9 and PRR7 compensate circadian period between 22°C and 28°C in shoot, not in root. The circadian rhythms of PRR9 or PRR7 transcript accumulation showed no difference at 22°C and 28°C in shoot, but differences were observed in root. In summary, our results reveal the specificity of dynamic circadian protein-protein interactions in organ-autonomous clocks and the critical roles of PRR9 and PRR7 in mechanisms regulating temperature compensation in aerial shoot system.

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. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.

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. Further genetic analysis shows COR28 is essential for the circadian function of PRR9 and PRR7. Together, our results support a role of COR27 and COR28 as nighttime repressors integrating circadian clock and plant cold stress responses.

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. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.

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. We also identified that both LNK1 and LNK2 recruitment to the evening element of PRR5 promoter via LNK1-RVE8 or LNK2-RVE8 proteins complex through electrophoretic mobility shift assay. Therefore LNK1 and LNK2 function as coactivator of dawn-phased MYB-like transcription factors, such as RVE8 in morning complex to regulate the target genes expression.

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. The two B. rapa GI alleles each fully rescued the delayed flowering of Arabidopsis gi-201 but showed differential rescue of perturbations in red light inhibition of hypocotyl elongation and altered cold and salt tolerance. The B. rapa R500 GI allele, which failed to rescue the hypocotyl and abiotic stress phenotypes, disrupted circadian period determination in Arabidopsis. Analysis of chimeric B. rapa GI alleles identified the causal nucleotide polymorphism, which results in an amino acid substitution (S264A) between the two GI proteins. This polymorphism underlies variation in circadian period, cold and salt tolerance, and red light inhibition of hypocotyl elongation. Loss-of-function mutations of B. rapa GI confer delayed flowering, perturbed circadian rhythms in leaf movement, and increased freezing and increased salt tolerance, consistent with effects of similar mutations in Arabidopsis. Collectively, these data suggest that allelic variation of GI-and possibly of clock genes in general-offers an attractive target for molecular breeding for enhanced stress tolerance and potentially for improved crop yield.

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. Mutational disruption of LNK1 and LNK2 function prevents transcriptional activation of PRR5 by RVE8. The LNKs lack known DNA binding domains, yet LNK1 acts as a transcriptional activator in yeast and in planta. Chromatin immunoprecipitation shows that LNK1 is recruited to the PRR5 and TOC1 promoters in planta. We conclude that LNK1 is a transcriptional coactivator necessary for expression of the clock genes PRR5 and TOC1 through recruitment to their promoters via interaction with bona fide DNA binding proteins such as RVE4 and RVE8.