ABSTRACT
BACKGROUND: Recent studies demonstrated that microbiota inhabiting the plant rhizosphere exhibit diel changes in abundance. To investigate the impact of plant circadian rhythms on bacterial and fungal rhythms in the rhizosphere, we analysed temporal changes in fungal and bacterial communities in the rhizosphere of Arabidopsis plants overexpressing or lacking function of the circadian clock gene LATE ELONGATED HYPOCOTYL (LHY). RESULTS: Under diel light-dark cycles, the knock-out mutant lhy-11 and the gain-of-function mutant lhy-ox both exhibited gene expression rhythms with altered timing and amplitude compared to wild-type plants. Distinct sets of bacteria and fungi were found to display rhythmic changes in abundance in the rhizosphere of both of these mutants, suggesting that abnormal patterns of rhythmicity in the plant host caused temporal reprogramming of the rhizosphere microbiome. This was associated with changes in microbial community structure, including changes in the abundance of fungal guilds known to impact on plant health. Under constant environmental conditions, microbial rhythmicity persisted in the rhizosphere of wild-type plants, indicating control by a circadian oscillator. In contrast, loss of rhythmicity in lhy-ox plants was associated with disrupted rhythms for the majority of rhizosphere microbiota. CONCLUSIONS: These results show that aberrant function of the plant circadian clock is associated with altered rhythmicity of rhizosphere bacteria and fungi. In the long term, this leads to changes in composition of the rhizosphere microbiome, with potential consequences for plant health. Further research will be required to understand the functional implications of these changes and how they impact on plant health and productivity.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Microbiota , Arabidopsis Proteins/genetics , Circadian Rhythm/genetics , Rhizosphere , Transcription Factors/genetics , Gene Expression Regulation, Plant , DNA-Binding Proteins/genetics , Arabidopsis/genetics , Arabidopsis/metabolismABSTRACT
Chromatin immunoprecipitation, or ChIP, is a powerful experimental technique for probing protein-DNA interactions in vivo. This assay can be used to investigate the association of a protein of interest with specific target loci. Alternatively, it can be combined with high-throughput sequencing technology to identify genome-wide binding sites. Here, we describe a ChIP protocol that was optimized for low-abundance transcription factors in Arabidopsis, and provide guidance on how to adapt it for other types of plants and proteins.
Subject(s)
Arabidopsis , Circadian Clocks , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Binding Sites , CLOCK Proteins , Chromatin , Chromatin ImmunoprecipitationABSTRACT
Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.
Subject(s)
Circadian Clocks , Medicago truncatula , Sinorhizobium meliloti , Cysteine/metabolism , Gene Expression Regulation, Plant , Medicago truncatula/metabolism , Nitrogen Fixation/physiology , Peptides/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Root Nodulation/genetics , Root Nodules, Plant/metabolism , SymbiosisABSTRACT
The LATE ELONGATED HYPOCOTYL (LHY) transcription factor functions as part of the oscillatory mechanism of the Arabidopsis circadian clock. This paper reports the genome-wide analysis of its binding targets and reveals a role in the control of abscisic acid (ABA) biosynthesis and downstream responses. LHY directly repressed expression of 9-cis-epoxycarotenoid dioxygenase enzymes, which catalyse the rate-limiting step of ABA biosynthesis. This suggested a mechanism for the circadian control of ABA accumulation in wild-type plants. Consistent with this hypothesis, ABA accumulated rhythmically in wild-type plants, peaking in the evening. LHY-overexpressing plants had reduced levels of ABA under drought stress, whereas loss-of-function mutants exhibited an altered rhythm of ABA accumulation. LHY also bound the promoter of multiple components of ABA signalling pathways, suggesting that it may also act to regulate responses downstream of the hormone. LHY promoted expression of ABA-responsive genes responsible for increased tolerance to drought and osmotic stress but alleviated the inhibitory effect of ABA on seed germination and plant growth. This study reveals a complex interaction between the circadian clock and ABA pathways, which is likely to make an important contribution to plant performance under drought and osmotic stress conditions.
Subject(s)
Abscisic Acid/biosynthesis , Arabidopsis/genetics , Arabidopsis/metabolism , Biosynthetic Pathways , Circadian Rhythm , DNA-Binding Proteins/metabolism , Genome, Plant , Signal Transduction , Transcription Factors/metabolism , Abscisic Acid/pharmacology , Arabidopsis/drug effects , Base Sequence , Binding Sites , Biosynthetic Pathways/drug effects , Circadian Clocks/drug effects , Circadian Clocks/genetics , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Gene Expression Regulation, Plant/drug effects , Gene Ontology , Promoter Regions, Genetic , Protein Binding/drug effectsABSTRACT
In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).
Subject(s)
Arabidopsis/physiology , Calcium/metabolism , Circadian Clocks/physiology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Calcium/physiology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Transcription Factors/metabolismABSTRACT
The network structure of the plant circadian clock is complex and direct regulatory interactions between individual components have proven particularly difficult to predict from genetic analyses. Here, we systematically investigate in vivo binding interactions between the morning-specific transcription factor, LATE ELONGATED HYPOCOTYL (LHY) and the promoters of other components of the network. We then demonstrate the functionality of these interactions by testing the responsiveness of the target gene to an ethanol-induced change in expression level of the LHY protein. We uncover novel, negative autoregulatory feedback loops from LHY and the closely related CIRCADIAN CLOCK ASSOCIATED-1 (CCA1) onto their own and each other's expression. Furthermore we show that LHY acts as a repressor of all other clock components, including PSEUDO-RESPONSE REGULATORs (PRRs) 9 and 7, which were previously thought to be positive regulatory targets. These experimental results lead to a substantial revision of the morning loops of the clock.
Subject(s)
Arabidopsis/genetics , Circadian Clocks/genetics , Circadian Rhythm/genetics , Gene Expression Regulation, Plant/genetics , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Transcription Factors/geneticsABSTRACT
Extremes of temperatures, drought and salinity cause widespread crop losses throughout the world and impose severe limitations on the amount of land that can be used for agricultural purposes. Hence, there is an urgent need to develop crops that perform better under such abiotic stress conditions. Here, we discuss intriguing, recent evidence that circadian clock contributes to plants' ability to tolerate different types of environmental stress, and to acclimate to them. The clock controls expression of a large fraction of abiotic stress-responsive genes, as well as biosynthesis and signaling downstream of stress response hormones. Conversely, abiotic stress results in altered expression and differential splicing of the clock genes, leading to altered oscillations of downstream stress-response pathways. We propose a range of mechanisms by which this intimate coupling between the circadian clock and environmental stress-response pathways may contribute to plant growth and survival under abiotic stress.
ABSTRACT
With fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.
Subject(s)
Chlorophyta/genetics , Gene Targeting/methods , Homologous Recombination , Algal Proteins/genetics , Ferritins/genetics , Gene Knock-In Techniques , Gene Knockout Techniques , Genes, Reporter , Genome, Plant , Luciferases/genetics , Nitrate Reductase/genetics , Transformation, GeneticABSTRACT
The plant circadian clock controls many aspects of growth and development, allowing an individual to adapt its physiology and metabolism in anticipation of diurnal and seasonal environmental changes. Circadian regulation of hormone levels and hormonal signalling modulates many features of development, including daily growth patterns and the breaking of seed dormancy. The clock also plays a role in seasonal day-length perception, allowing plants to optimally time key development transitions, such as reproduction. Moreover, the clock restricts (gates) the sensitivity of a plant's response to environmental cues, such as light and stress, to specific times of the day, ensuring that the plant can distinguish between normal fluctuations and longer-term changes. The central oscillator controls many of these output pathways via rhythmic gene expression, with several of the core clock components encoding transcription factors. Post-transcriptional processes are also likely to make an important contribution to the circadian regulation of output pathways. The plant circadian clock plays a role in regulating fitness, hybrid vigour and numerous stress responses. Thus elucidating the complexities of the circadian output mechanisms and their regulation may provide new avenues for crop enhancement.
Subject(s)
Circadian Clocks/physiology , Circadian Rhythm/physiology , Plant Development , Plant Physiological Phenomena , Arabidopsis/genetics , Arabidopsis/physiology , Circadian Clocks/genetics , Circadian Rhythm/genetics , Circadian Rhythm Signaling Peptides and Proteins/genetics , Circadian Rhythm Signaling Peptides and Proteins/physiology , Gene Expression Regulation , Photoperiod , Plant Growth Regulators/physiologyABSTRACT
Photoperiod sensors allow physiological adaptation to the changing seasons. The prevalent hypothesis is that day length perception is mediated through coupling of an endogenous rhythm with an external light signal. Sufficient molecular data are available to test this quantitatively in plants, though not yet in mammals. In Arabidopsis, the clock-regulated genes CONSTANS (CO) and FLAVIN, KELCH, F-BOX (FKF1) and their light-sensitive proteins are thought to form an external coincidence sensor. Here, we model the integration of light and timing information by CO, its target gene FLOWERING LOCUS T (FT), and the circadian clock. Among other predictions, our models show that FKF1 activates FT. We demonstrate experimentally that this effect is independent of the known activation of CO by FKF1, thus we locate a major, novel controller of photoperiodism. External coincidence is part of a complex photoperiod sensor: modeling makes this complexity explicit and may thus contribute to crop improvement.
Subject(s)
Arabidopsis/physiology , Flowers/physiology , Gene Expression Regulation, Plant , Models, Genetic , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biological Clocks , DNA-Binding Proteins/genetics , Gene Regulatory Networks , Photoperiod , Transcription Factors/geneticsABSTRACT
The transcriptional regulation of the LATE ELONGATED HYPOCOTYL (LHY) gene is key to the structure of the circadian oscillator, integrating information from multiple regulatory pathways. We identified a minimal region of the LHY promoter that was sufficient for rhythmic expression. Another upstream sequence was also required for appropriate waveform of transcription and for maximum amplitude of oscillations under both diurnal and free-running conditions. We showed that two classes of protein complexes interact with a G-box and with novel 5A motifs; mutation of these sites reduced the amplitude of oscillation and broadened the peak of expression. A genome-wide bioinformatic analysis showed that these sites were enriched in phase-specific clusters of rhythmically expressed genes. Comparative genomic analyses showed that these motifs were conserved in orthologous promoters from several species. A position-specific scoring matrix for the 5A sites suggested similarity to CArG boxes, which are recognized by MADS box transcription factors. In support of this, the FLOWERING LOCUS C (FLC) protein was shown to interact with the LHY promoter in planta. This suggests a mechanism by which FLC might affect circadian period.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Circadian Rhythm , Promoter Regions, Genetic , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Binding Sites , Chromosome Mapping , Comparative Genomic Hybridization , Conserved Sequence , DNA Mutational Analysis , DNA, Plant/genetics , Gene Deletion , Gene Expression Regulation, Plant , Photoperiod , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Position-Specific Scoring Matrices , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Multiple photoreceptors contribute to the entrainment of the Arabidopsis circadian clock to daily cycles of light and darkness but little is known of the mechanisms by which these pathways affect the central oscillator. Here we investigate the epistatic interaction between DE-ETIOLATED 1 (DET1), a negative regulator of light-regulated gene expression, and LATE ELONGATED HYPOCOTYL (LHY), one of the core components of the circadian oscillator. The daily onset of LHY gene expression was advanced by approximately 4 h in det1-1 mutant plants, suggesting that the wild-type DET1 protein might function to repress its transcription during the subjective night. lhy-1 det1-1 double mutants exhibited arrhythmic expression of the CAB gene in constant light, similar to the lhy-1 mutant parent. However, additive effects of the lhy-1 and det1-1 mutations on CAB2 expression patterns were revealed under diurnal light-dark cycles. Since the lhy-1 mutation causes aberrant, constitutive transcription of LHY from a constitutive viral promoter, this observation indicated that effects of DET1 were not mediated through the regulation of LHY transcription. Furthermore, the light-driven, rhythmic accumulation of the LHY protein in the lhy-1 mutant was altered by the det1-1 mutation, suggesting that DET1 might regulate LHY expression at the post-transcriptional level. In vitro protein degradation assays demonstrated that the LHY protein is turned over rapidly through the proteasome pathway. Similar degradation was observed whether plant tissue was harvested during the light or dark portion of the diurnal cycle, but the process was significantly accelerated in det1-1 mutant extracts. These results indicate that the wild-type DET1 protein acts to inhibit the proteolytic turnover of the LHY protein, and suggest a mechanism for the period-shortening effect of the det1-1 mutation. These findings add to recent evidence suggesting a role for DET1 in a ubiquitination pathway and identify a substrate for DET1-regulated protein turn-over.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Circadian Rhythm/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Chlorophyll Binding Proteins , Cysteine Proteinase Inhibitors/pharmacology , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental/radiation effects , Gene Expression Regulation, Plant/radiation effects , Genotype , Immunoblotting , Intracellular Signaling Peptides and Proteins , Leupeptins/pharmacology , Light , Light-Harvesting Protein Complexes/genetics , Luciferases/genetics , Luciferases/metabolism , Mutation , Nuclear Proteins/genetics , Photosystem II Protein Complex/genetics , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Proteasome Inhibitors , RNA, Plant/genetics , RNA, Plant/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Transcription Factors/geneticsABSTRACT
The transcription factor LHY and the related protein CCA1 perform overlapping functions in a regulatory feedback loop that is closely associated with the circadian oscillator of Arabidopsis: Overexpression of LHY abolished function of the circadian clock in constant light, but rhythmic expression of several circadian clock-regulated transcripts was observed under light- dark cycles. These oscillations correlated with high amplitude changes in LHY protein levels, caused by light-induced translation of the LHY transcript. Increases in LHY protein levels were also observed in light-grown wild-type plants, when light signals coincided with the circadian-regulated peak of LHY transcription at dawn. Unexpectedly, translational induction coincided with acute downregulation of LHY transcript levels. We suggest that the simultaneous translational induction and transcriptional repression of LHY expression play a role to narrow the peak of LHY protein synthesis at dawn and increase the robustness and accuracy of circadian oscillations. Strong phase shifting responses to light signals were observed in plants lacking function of LHY, CCA1 or both, suggesting that light-regulated expression of these proteins does not mediate entrainment of the clock to light-dark cycles.
Subject(s)
Arabidopsis/physiology , DNA-Binding Proteins/genetics , Light , Protein Biosynthesis/physiology , Transcription Factors/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Circadian Rhythm/genetics , Circadian Rhythm/physiology , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Plant/physiology , Transcription Factors/biosynthesisABSTRACT
Daylength, or photoperiod, is perceived as a seasonal signal for the control of flowering of many plants. The measurement of daylength is thought to be mediated through the interaction of phototransduction pathways with a circadian rhythm, so that flowering is induced (in long-day plants) or repressed (in short-day plants) when light coincides with a sensitive phase of the circadian cycle. To test this hypothesis in the facultative long-day plant, Arabidopsis thaliana, we used varying, non-24-hr light/dark cycles to alter the timing of circadian rhythms of gene expression relative to dawn and dusk. Effects on circadian rhythms were correlated with those on flowering times. We show that conditions that displaced subjective night events, such as expression of the flowering time regulator CONSTANS into the light portion of the cycle, were perceived as longer days. This work demonstrates that the perception of daylength in Arabidopsis relies on adjustments of the phase angle of circadian rhythms relative to the light/dark cycle, rather than on the measurement of the absolute duration of light and darkness.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Arabidopsis/physiology , DNA-Binding Proteins/genetics , Transcription Factors/genetics , Arabidopsis Proteins/physiology , Cell Division , Circadian Rhythm , DNA-Binding Proteins/physiology , Genes, Reporter , Light , Luciferases/metabolism , Plant Physiological Phenomena , Plants, Genetically Modified , RNA, Messenger/metabolism , Time Factors , Transcription Factors/physiologyABSTRACT
The LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1) genes encode closely related MYB transcription factors, which regulate circadian rhythms in Arabidopsis. LHY and CCA1 verify some of the properties of oscillator components, since (i) expression of their transcripts and protein exhibits circadian oscillations; (ii) their constitutive expression abolishes overt rhythmicity and (iii) they function as components of a negative transcriptional feedback loop. LHY and CCA1 have been proposed to function in conjunction with the pseudo response regulator TOC1, as components of the circadian oscillator. The regulation of their respective transcripts and protein levels in response to light signals suggests that these proteins may also mediate the regulation of circadian rhythms by light. This review discusses experimental evidence for these hypotheses.
Subject(s)
Arabidopsis/genetics , Circadian Rhythm/genetics , DNA-Binding Proteins/genetics , Plant Proteins/genetics , Proto-Oncogene Proteins c-myb , Transcription Factors/genetics , Amino Acid Sequence , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Circadian Rhythm/physiology , Darkness , Gene Expression Regulation, Plant , Light , Molecular Sequence Data , Mutation , Sequence Homology, Amino AcidABSTRACT
Several genes are known to regulate circadian rhythms in Arabidopsis, but the identity of the central oscillator has not been established. LHY and CCA1 are related MYB-like transcription factors proposed to be closely involved. Here we demonstrate that, as shown previously for CCA1, inactivation of LHY shortens the period of circadian rhythms in gene expression and leaf movements. By constructing lhy cca1-1 double mutants, we show that LHY and CCA1 are partially redundant and essential for the maintenance of circadian rhythms in constant light. Under light/dark cycles the lhy cca1-1 plants show dramatically earlier phases of expression of GI and TOC1, genes associated with the generation of circadian rhythms and the promotion of LHY and CCA1 expression. We conclude that LHY and CCA1 appear to be negative regulatory elements required for central oscillator function.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Circadian Rhythm/genetics , DNA-Binding Proteins/genetics , Genes, Plant , Transcription Factors/genetics , Alleles , Arabidopsis/growth & development , Arabidopsis/physiology , Circadian Rhythm/physiology , Gene Expression Regulation, Plant , Hypocotyl/growth & development , Models, Biological , Movement , Mutation , Phenotype , Photoperiod , Plant Leaves/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolismABSTRACT
The EARLY FLOWERING 3 (ELF3) gene of Arabidopsis regulates plant morphology, flowering time and circadian rhythms. ELF3 was proposed to function as a modulator of light signal transduction downstream of phytochromes, and, perhaps, other photoreceptors. Recent work indicates that ELF3 encodes a novel nuclear protein that is expressed rhythmically and interacts with phytochrome B. How ELF3 mediates the circadian gating of light responses and regulates light input to the clock is the subject of discussion.