Crosstalk between the circadian clock and innate immunity in Arabidopsis.
ABSTRACT: 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. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.
Project description:The circadian clock drives daily rhythms of many plant physiological responses, providing a competitive advantage that improves plant fitness and survival rates [1-5]. Whereas multiple environmental cues are predicted to regulate the plant clock function, most studies focused on understanding the effects of light and temperature [5-8]. Increasing evidence indicates a significant role of plant-pathogen interactions on clock regulation [9, 10], but the underlying mechanisms remain elusive. In Arabidopsis, the clock function largely relies on a transcriptional feedback loop between morning (CCA1 and LHY)- and evening (TOC1)-expressed transcription factors [6-8]. Here, we focused on these core components to investigate the Arabidopsis clock regulation using a unique biotic stress approach. We found that a single-leaf Pseudomonas syringae infection systemically lengthened the period and reduced the amplitude of circadian rhythms in distal uninfected tissues. Remarkably, the low-amplitude phenotype observed upon infection was recapitulated by a transient treatment with the defense-related phytohormone salicylic acid (SA), which also triggered a significant clock phase delay. Strikingly, despite SA-modulated circadian rhythms, we revealed that the master regulator of SA signaling, NPR1 [11, 12], antagonized clock responses triggered by both SA treatment and P. syringae. In contrast, we uncovered that the NADPH oxidase RBOHD  largely mediated the aforementioned clock responses after either SA treatment or the bacterial infection. Altogether, we demonstrated novel and unexpected roles for SA, NPR1, and redox signaling in clock regulation by P. syringae and revealed a previously unrecognized layer of systemic clock regulation by locally perceived environmental cues.
Project description:The circadian clock is an endogenous 24?h oscillator regulating many critical biological processes in plants. One of the key characteristics of the circadian clock is that it is buffered against temperature, maintaining an approximately 24?h rhythm over a broad physiological temperature range. Here, we tested temperature-buffering capacity of the circadian clock across a number of Arabidopsis accessions using several circadian clock reporters: leaf movement, CCA1:LUC and LHY:LUC. We found that leaf movement was the best temperature buffered circadian output. On the other hand, when temperature increases, circadian rhythms of CCA1 and LHY transcription shorten considerably across all accessions, indicating that the clock driving expression of CCA1 and LHY is not perfectly buffered. This feature might be crucial to plants growing in a constantly changing environment, and here, we provide insight into the importance of period shortening to plant growth performance and the benefits of a flexible clock.
Project description:The C-Repeat Binding Factor (CBF) cold-response pathway has a prominent role in cold acclimation, the process whereby certain plants increase tolerance to freezing in response to low nonfreezing temperatures. In Arabidopsis, the CBF pathway is characterized by rapid induction of the C-Repeat Binding Factor 1 (CBF1), CBF2, and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the "CBF regulon." Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2, and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and ChIP experiments indicating that both these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., Circadian Clock-Associated 1 (CCA1) and Late Elongated Hypocotyl (LHY). In plants carrying the cca1-11/lhy-21 double mutation, cold induction of CBF1, CBF2, and CBF3 was greatly impaired, and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, although with significantly reduced amplitude. Circadian regulation and cold induction of three CBF regulon genes, i.e., COld-regulated Gene15a (COR15A), COR47, and COR78, also were greatly diminished in plants carrying the cca1-11/lhy-21 double mutation. Furthermore, the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance in both nonacclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold-response pathway.
Project description:Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.
Project description:BACKGROUND: Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. RESULTS: We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions. CONCLUSIONS: Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla  indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism.
Project description:BACKGROUND: Plant circadian clocks regulate many photoperiodic and diurnal responses that are conserved among plant species. The plant circadian clock system has been uncovered in the model plant, Arabidopsis thaliana, using genetics and systems biology approaches. However, it is still not clear how the clock system had been organized in the evolutionary history of plants. We recently revealed the molecular phylogeny of LHY/CCA1 genes, one of the essential components of the clock system. The aims of this study are to reconstruct the phylogenetic relationships of angiosperm clock-associated PRR genes, the partner of the LHY/CCA1 genes, and to clarify the evolutionary history of the plant clock system in angiosperm lineages. RESULTS: In the present study, to investigate the molecular phylogeny of PRR genes, we performed two approaches: reconstruction of phylogenetic trees and examination of syntenic relationships. Phylogenetic analyses revealed that PRR genes had diverged into three clades prior to the speciation of monocots and eudicots. Furthermore, copy numbers of PRR genes have been independently increased in monocots and eudicots as a result of ancient chromosomal duplication events. CONCLUSIONS: Based on the molecular phylogenies of both PRR genes and LHY/CCA1 genes, we inferred the evolutionary process of the plant clock system in angiosperms. This scenario provides evolutionary information that a common ancestor of monocots and eudicots had retained the basic components required for reconstructing a clock system and that the plant circadian clock may have become a more elaborate mechanism after the speciation of monocots and eudicots because of the gene expansion that resulted from polyploidy events.
Project description:Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.
Project description:In Arabidopsis, the circadian clock central oscillator genes are important cellular components to generate and maintain circadian rhythms. There is a negative feedback loop between the morning expressed CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and evening expressed TOC1 (TIMING OF CAB EXPRESSION 1). CCA1 and LHY negatively regulate the expression of TOC1, while TOC1 also binds to the promoters of CCA1 and LHY to repress their expression. Recent studies indicate that histone modifications play an important role in the regulation of the central oscillators. However, the regulatory relationship between histone modifications and the circadian clock genes remains largely unclear. In this study, we found that the Lysine-Specific Demethylase 1 (LSD1)-like histone demethylases, LDL1 and LDL2, can interact with CCA1/LHY to repress the expression of TOC1. ChIP-Seq analysis indicated that LDL1 targets a subset of genes involved in the circadian rhythm regulated by CCA1. Furthermore, LDL1 and LDL2 interact with the histone deacetylase HDA6 and co-regulate TOC1 by histone demetylation and deacetylaion. These results provide new insight into the molecular mechanism of how the circadian clock central oscillator genes are regulated through histone modifications.
Project description:The circadian clock provides robust, ?24 hr biological rhythms throughout the eukaryotes. The clock gene circuit in plants comprises interlocking transcriptional feedback loops, reviewed in , whereby the morning-expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of evening genes, notably TIMING OF CAB EXPRESSION 1 (TOC1). EARLY FLOWERING 3 (ELF3) has been implicated as a repressor of light signaling to the clock [2, 3] and, paradoxically, as an activator of the light-induced genes CCA1 and LHY [4, 5]. We use cca1-11 lhy-21 elf3-4 plants to separate the repressive function of ELF3 from its downstream targets CCA1 and LHY. We further demonstrate that ELF3 associates physically with the promoter of PSEUDO-RESPONSE REGULATOR 9 (PRR9), a repressor of CCA1 and LHY expression, in a time-dependent fashion. The repressive function of ELF3 is thus consistent with indirect activation of LHY and CCA1, in a double-negative connection via a direct ELF3 target, PRR9. This mechanism reconciles the functions of ELF3 in the clock network during the night and points to further effects of ELF3 during the day.
Project description:In Arabidopsis, the circadian rhythm is associated with multiple important biological processes and maintained by multiple interconnected loops that generate robust rhythms. The circadian clock central loop is a negative feedback loop composed of the core circadian clock components. TOC1 (TIMING OF CAB EXPRESSION 1) is highly expressed in the evening and negatively regulates the expression of CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL). CCA1/LHY also binds to the promoter of TOC1 and represses the TOC1 expression. Our recent research revealed that the histone modification complex comprising of LYSINE-SPECIFIC DEMETHYLASE 1 (LSD1)-LIKE 1/2 (LDL1/2) and HISTONE DEACETYLASE 6 (HDA6) can be recruited by CCA1/LHY to repress TOC1 expression. In this study, we found that HDA6, LDL1, and LDL2 can interact with TOC1, and the LDL1/2-HDA6 complex is associate with TOC1 to repress the CCA1/LHY expression. Furthermore, LDL1/2-HDA6 and TOC1 co-target a subset of genes involved in the circadian rhythm. Collectively, our results indicate that the LDL1/2-HDA6 histone modification complex is important for the regulation of the core circadian clock components.