RESUMO
Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3' untranslated regions is associated with decreased relative transcript abundance and defective RNA 3' end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.
Assuntos
Adenosina/análogos & derivados , Arabidopsis/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Análise de Sequência de RNA , Adenosina/metabolismo , Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Metilação , Nanoporos , Poli A/genética , Poli A/metabolismo , Capuzes de RNA , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Plantas/química , RNA de Plantas/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genéticaRESUMO
Individual plant cells have a genetic circuit, the circadian clock, that times key processes to the day-night cycle. These clocks are aligned to the day-night cycle by multiple environmental signals that vary across the plant. How does the plant integrate clock rhythms, both within and between organs, to ensure coordinated timing? To address this question, we examined the clock at the sub-tissue level across Arabidopsis thaliana seedlings under multiple environmental conditions and genetic backgrounds. Our results show that the clock runs at different speeds (periods) in each organ, which causes the clock to peak at different times across the plant in both constant environmental conditions and light-dark (LD) cycles. Closer examination reveals that spatial waves of clock gene expression propagate both within and between organs. Using a combination of modeling and experiment, we reveal that these spatial waves are the result of the period differences between organs and local coupling, rather than long-distance signaling. With further experiments we show that the endogenous period differences, and thus the spatial waves, can be generated by the organ specificity of inputs into the clock. We demonstrate this by modulating periods using light and metabolic signals, as well as with genetic perturbations. Our results reveal that plant clocks can be set locally by organ-specific inputs but coordinated globally via spatial waves of clock gene expression.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Redes Reguladoras de Genes , Especificidade de Órgãos/genética , Fotoperíodo , Plântula/genética , Plântula/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The Arabidopsis circadian clock orchestrates gene regulation across the day/night cycle. Although a multiple feedback loop circuit has been shown to generate the 24-hr rhythm, it remains unclear how robust the clock is in individual cells, or how clock timing is coordinated across the plant. Here we examine clock activity at the single cell level across Arabidopsis seedlings over several days under constant environmental conditions. Our data reveal robust single cell oscillations, albeit desynchronised. In particular, we observe two waves of clock activity; one going down, and one up the root. We also find evidence of cell-to-cell coupling of the clock, especially in the root tip. A simple model shows that cell-to-cell coupling and our measured period differences between cells can generate the observed waves. Our results reveal the spatial structure of the plant clock and suggest that unlike the centralised mammalian clock, the Arabidopsis clock has multiple coordination points.
Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Relógios Circadianos , Regulação da Expressão Gênica de Plantas , Células , Redes Reguladoras de Genes , PlântulaRESUMO
Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphoenolpyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal CO2 fixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the transcript abundance of many core circadian clock genes, suggesting that perturbing the only known link between CAM and the circadian clock feeds back to perturb the central circadian clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, malate accumulation, CAM productivity, and core circadian clock robustness.
Assuntos
Relógios Circadianos/fisiologia , Fosfoenolpiruvato Carboxilase/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica de Plantas , Fosfoenolpiruvato Carboxilase/genética , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Relógios Circadianos/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Raízes de Plantas/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Gravitropismo , Ácidos Indolacéticos/metabolismo , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Over the last two decades, the development of high-throughput techniques has enabled us to probe the plant circadian clock, a key coordinator of vital biological processes, in ways previously impossible. With the circadian clock increasingly implicated in key fitness and signalling pathways, this has opened up new avenues for understanding plant development and signalling. Our tool-kit has been constantly improving through continual development and novel techniques that increase throughput, reduce costs and allow higher resolution on the cellular and subcellular levels. With circadian assays becoming more accessible and relevant than ever to researchers, in this paper we offer a review of the techniques currently available before considering the horizons in circadian investigation at ever higher throughputs and resolutions.
RESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Proteínas de Ligação a DNA/metabolismo , Temperatura , Fatores de Transcrição/metabolismo , Análise de Variância , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fatores de Transcrição/genéticaRESUMO
Correct circadian regulation increases plant productivity, and photosynthesis is circadian-regulated. Here, we discuss the regulatory basis for the circadian control of photosynthesis. We discuss candidate mechanisms underpinning circadian oscillations of light harvesting and consider how the circadian clock modulates CO2 fixation by Rubisco. We show that new techniques may provide a platform to better understand the signalling pathways that couple the circadian clock with the photosynthetic apparatus. Finally, we discuss how understanding circadian regulation in model systems is underpinning research into the impact of circadian regulation in crop species.
Assuntos
Ritmo Circadiano/fisiologia , Produtos Agrícolas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Fotossíntese/fisiologia , Dióxido de Carbono/metabolismo , Cloroplastos/genética , Produtos Agrícolas/metabolismo , Luz , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
The identification and cloning of full-length homologs of circadian clock genes from Picea abies represent a first step to study the function and evolution of the circadian clock in gymnosperms. Phylogenetic analyses suggest that the sequences of key circadian clock genes are conserved between angiosperms and gymnosperms. though fewer homologous copies were found for most gene families in P. abies. We detected diurnal cycling of circadian clock genes in P. abies using quantitative real-time PCR; however, cycling appeared to be rapidly dampened under free-running conditions. Given the unexpected absence of transcriptional cycling during constant conditions, we employed a complementary method to assay circadian rhythmic outputs and measured delayed fluorescence in seedlings of Norway spruce. Neither of the two approaches to study circadian rhythms in Norway spruce could detect robust â¼24 h cycling behavior under constant conditions. These data suggest gene conservation but fundamental differences in clock function between gymnosperms and other plant taxa.
Assuntos
Ritmo Circadiano/fisiologia , Picea/genética , Picea/fisiologia , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , Filogenia , Picea/classificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Estimation of the period length of time-course data from cyclical biological processes, such as those driven by the circadian pacemaker, is crucial for inferring the properties of the biological clock found in many living organisms. We propose a methodology for period estimation based on spectrum resampling (SR) techniques. Simulation studies show that SR is superior and more robust to non-sinusoidal and noisy cycles than a currently used routine based on Fourier approximations. In addition, a simple fit to the oscillations using linear least squares is available, together with a non-parametric test for detecting changes in period length which allows for period estimates with different variances, as frequently encountered in practice. The proposed methods are motivated by and applied to various data examples from chronobiology.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Interpretação Estatística de Dados , Análise de Regressão , Animais , Arabidopsis/crescimento & desenvolvimento , Cromoterapia/métodos , Neoplasias Colorretais/tratamento farmacológico , Simulação por Computador , Humanos , Pneumopatias/tratamento farmacológico , Camundongos , Proteínas Circadianas Period/uso terapêutico , Temperatura Cutânea/efeitos dos fármacosRESUMO
Circadian clocks exhibit 'temperature compensation', meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature-dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature-compensated clock model by adding passive temperature effects into only the light-sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature-dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems-level understanding of period control in the plant circadian oscillator.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Modelos Biológicos , Proteínas de Arabidopsis/genética , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Modelos Teóricos , Mutação , Plantas Geneticamente Modificadas , Transdução de Sinais , Temperatura , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3' end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3' tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3' tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3' tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3' tagging, ribosome clearance, and transcript degradation.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Processamento de Terminações 3' de RNA , Ribossomos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Sequência de Bases , Genes Fúngicos , Humanos , Modelos Biológicos , Mutação , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismoRESUMO
The plant circadian clock plays an important role in enhancing performance and increasing vegetative yield. Much of our current understanding of the mechanism and function of the plant clock has come from the development of Arabidopsis thaliana as a model circadian organism. Key to this rapid progress has been the development of robust circadian markers, specifically circadian-regulated luciferase reporter genes. Studies of the clock in crop species and non-model organisms are currently hindered by the absence of a simple high-throughput universal assay for clock function, accuracy and robustness. Delayed fluorescence (DF) is a fundamental process occurring in all photosynthetic organisms. It is luminescence-produced post-illumination due to charge recombination in photosystem II (PSII) leading to excitation of P680 and the subsequent emission of a photon. Here we report that the amount of DF oscillates with an approximately 24-h period and is under the control of the circadian clock in a diverse selection of plants. Thus, DF provides a simple clock output that may allow the clock to be assayed in vivo in any photosynthetic organism. Furthermore, our data provide direct evidence that the nucleus-encoded, three-loop circadian oscillator underlies rhythms of PSII activity in the chloroplast. This simple, high-throughput and non-transgenic assay could be integrated into crop breeding programmes, the assay allows the selection of plants that have robust and accurate clocks, and possibly enhanced performance and vegetative yield. This assay could also be used to characterize rapidly the role and function of any novel Arabidopsis circadian mutant.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Fluorescência , Magnoliopsida/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Cloroplastos/fisiologia , Fotoperíodo , Complexo de Proteína do Fotossistema II/fisiologia , Regiões Promotoras GenéticasRESUMO
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.
Assuntos
Arabidopsis/fisiologia , Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Retroalimentação/fisiologia , Modelos Teóricos , Previsões/métodos , Modelos Biológicos , Fotoperíodo , Projetos de PesquisaRESUMO
Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27 degrees C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 12 degrees C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27 degrees C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 12 degrees C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 17 degrees C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.