RESUMO
Phase separation is an important mechanism for regulating various cellular functions. The EARLY FLOWERING 3 (ELF3) protein, an essential element of the EVENING COMPLEX (EC) involved in circadian clock regulation, has been shown to undergo phase separation. ELF3 is known to significantly influence elongation growth and flowering time regulation, and this is postulated to be due to whether the protein is in the dilute or phase-separated state. Here, we present a brief overview of methods for analyzing ELF3 phase separation in vitro, including the generation of phase diagrams as a function of pH and salt versus protein concentrations, optical microscopy, fluorescence recovery after photobleaching (FRAP), and turbidity assays.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Separação de Fases , Mutação , Luz , Relógios Circadianos/fisiologia , Regulação da Expressão Gênica de Plantas , Ritmo Circadiano/fisiologiaRESUMO
Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.
Assuntos
Proteínas de Ligação a DNA , DNA , Ensaio de Desvio de Mobilidade Eletroforética , Temperatura , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , DNA/química , Eletroforese em Gel de PoliacrilamidaRESUMO
RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dynamic and subject to rapid changes in response to environmental cues, temperature in particular. Thermosensitive RNA secondary structures have been harnessed by multiple organisms to survey their temperature environment and to adjust gene expression accordingly. It is thus highly desirable to observe RNA structural changes in real time over a range of temperatures. Multiple approaches have been developed to study structural dynamics, but many of these require extensive processing of the RNA, large amounts of RNA input, and/or cannot be applied under physiological conditions. Here, we describe the use of a dually fluorescently labeled RNA oligonucleotide (containing a predicted hairpin structure) to monitor subtle RNA structural dynamics in vitro by Förster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy. These approaches can be employed under physiologically relevant conditions over a range of temperatures and with RNA concentrations as low as 200 nM; they enable us to observe RNA structural dynamics in real time and to correlate these dynamics with changes in biological processes such as translation.
Assuntos
Transferência Ressonante de Energia de Fluorescência , RNA , RNA/química , Temperatura , Dicroísmo Circular , OligonucleotídeosRESUMO
BACKGROUND: Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in adapting barley and wheat to northern latitudes. How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. RESULTS: We find that phytochrome C (PHYC) is essential for flowering in Brachypodium distachyon. Conversely, ELF3 acts as a floral repressor and elf3 mutants display a constitutive long day phenotype and transcriptome. We find that ELF3 and PHYC occur in a common complex. ELF3 associates with the promoters of a number of conserved regulators of flowering, including PPD1 and VRN1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in short days and is necessary for rapid flowering in response to long days. PHYC is in the active Pfr state at the end of the day, but we observe it undergoes dark reversion over the course of the night. CONCLUSIONS: We propose that PHYC acts as a molecular timer and communicates information on night-length to the circadian clock via ELF3.
Assuntos
Brachypodium , Fitocromo , Fitocromo/genética , Fitocromo/metabolismo , Brachypodium/genética , Brachypodium/metabolismo , Fotoperíodo , Flores/genética , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Histones and transcription factors (TFs) are two important DNA-binding proteins that interact, compete, and together regulate transcriptional processes in response to diverse internal and external stimuli. Condition-specific depletion of histones in Saccharomyces cerevisiae using a galactose-inducible H3 promoter provides a suitable framework for examining transcriptional alteration resulting from reduced nucleosome content. However, the effect on DNA binding activities of TFs is yet to be fully explored. In this work, we combine ChIP-seq of H3 with RNA-seq to elucidate the genome-scale relationships between H3 occupancy patterns and transcriptional dynamics before and after global H3 depletion. ChIP-seq of Rap1 is also conducted in the H3-depletion and control treatments, to investigate the interplay between this master regulator TF and nucleosomal H3, and to explore the impact on diverse transcriptional responses of different groups of target genes and functions. Ultimately, we propose a working model and testable hypotheses regarding the impact of global and local H3 depletion on transcriptional changes. We also demonstrate different potential modes of interaction between Rap1 and H3, which sheds light on the potential multifunctional regulatory capabilities of Rap1 and potentially other pioneer factors.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
In 1998, Bill Gray and colleagues showed that warm temperatures trigger arabidopsis hypocotyl elongation in an auxin-dependent manner. This laid the foundation for a vibrant research discipline. With several active members of the 'thermomorphogenesis' community, we here reflect on 25 years of elevated ambient temperature research and look to the future.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Temperatura , Hipocótilo/metabolismo , Ácidos IndolacéticosRESUMO
Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Príons , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Temperature is a key environmental cue that influences the distribution and behavior of plants globally. Understanding how plants sense temperature and integrate this information into their development is important to determine how plants adapt to climate change and to apply this knowledge to the breeding of climate-resilient crops. The mechanisms of temperature perception in eukaryotes are only just beginning to be understood, with multiple molecular phenomena with inherent temperature dependencies, such as RNA melting, phytochrome dark reversion, and protein phase change, being exploited by nature to create thermosensory signaling networks. Here, we review recent progress in understanding how temperature sensing in four major pathways in Arabidopsis thaliana occurs: vernalization, cold stress, thermomorphogenesis, and heat stress. We discuss outstanding questions in the field and the importance of these mechanisms in the context of breeding climate-resilient crops.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Temperatura , Melhoramento Vegetal , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Produtos Agrícolas , Regulação da Expressão Gênica de PlantasRESUMO
We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.
Assuntos
Dióxido de Carbono , Mudança Climática , Estresse Fisiológico , Dióxido de Carbono/metabolismo , Transpiração Vegetal/fisiologia , Plantas/metabolismo , Água/metabolismoRESUMO
Plants use photoperiodism to activate flowering in response to a particular daylength. In rice, flowering is accelerated in short-day conditions, and even a brief exposure to light during the dark period (night-break) is sufficient to delay flowering. Although many of the genes involved in controlling flowering in rice have been uncovered, how the long- and short-day flowering pathways are integrated, and the mechanism of photoperiod perception is not understood. While many of the signaling components controlling photoperiod-activated flowering are conserved between Arabidopsis and rice, flowering in these two systems is activated by opposite photoperiods. Here we establish that photoperiodism in rice is controlled by the evening complex (EC). We show that mutants in the EC genes LUX ARRYTHMO (LUX) and EARLY FLOWERING3 (ELF3) paralogs abolish rice flowering. We also show that the EC directly binds and suppresses the expression of flowering repressors, including PRR37 and Ghd7. We further demonstrate that light acts via phyB to cause a rapid and sustained posttranslational modification of ELF3-1. Our results suggest a mechanism by which the EC is able to control both long- and short-day flowering pathways.
Assuntos
Flores , Oryza , Fotoperíodo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Flores/genética , Flores/crescimento & desenvolvimento , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Luz , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Plants show remarkable phenotypic plasticity and are able to adjust their morphology and development to diverse environmental stimuli. Morphological acclimation responses to elevated ambient temperatures are collectively termed thermomorphogenesis. In Arabidopsis thaliana, morphological changes are coordinated to a large extent by the transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which in turn is regulated by several thermosensing mechanisms and modulators. Here, we review recent advances in the identification of factors that regulate thermomorphogenesis of Arabidopsis seedlings by affecting PIF4 expression and PIF4 activity. We summarize newly identified thermosensing mechanisms and highlight work on the emerging topic of organ- and tissue-specificity in the regulation of thermomorphogenesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas/genética , Fitocromo/metabolismo , TemperaturaRESUMO
Light is a key determinant for plant growth, development, and ultimately yield. Phytochromes, red/far-red photoreceptors, play an important role in plant architecture, stress tolerance, and productivity. In the model plant Arabidopsis, it has been shown that PHYTOCHROME-INTERACTING FACTORS (PIFs; bHLH transcription factors) act as central hubs in the integration of external stimuli to regulate plant development. Recent studies have unveiled the importance of PIFs in crops. They are involved in the modulation of plant architecture and productivity through the regulation of cell division and elongation in response to different environmental cues. These studies show that different PIFs have overlapping but also distinct functions in the regulation of plant growth. Therefore, understanding the molecular mechanisms by which PIFs regulate plant development is crucial to improve crop productivity under both optimal and adverse environmental conditions. In this review, we discuss current knowledge of PIFs acting as integrators of light and other signals in different crops, with particular focus on the role of PIFs in responding to different environmental conditions and how this can be used to improve crop productivity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Fitocromo/genética , Fitocromo/metabolismo , Plantas/metabolismoRESUMO
Many plants are able to regenerate upon cutting, and this process can be enhanced in vitro by incubating explants on hormone-supplemented media. While such protocols have been used for decades, little is known about the molecular details of how incubation conditions influence their efficiency. In this study, we find that warm temperature promotes both callus formation and shoot regeneration in Arabidopsis thaliana. We show that such an increase in shoot regenerative capacity at higher temperatures correlates with the enhanced expression of several regeneration-associated genes, such as CUP-SHAPED COTYLEDON 1 (CUC1) encoding a transcription factor involved in shoot meristem formation and YUCCAs (YUCs) encoding auxin biosynthesis enzymes. ChIP-sequencing analyses further reveal that histone variant H2A.Z is enriched on these loci at 17°C, while its occupancy is reduced by an increase in ambient temperature to 27°C. Moreover, we provide genetic evidence to demonstrate that H2A.Z acts as a repressor of de novo shoot organogenesis since H2A.Z-depleted mutants display enhanced shoot regeneration. This study thus uncovers a new chromatin-based mechanism that influences hormone-induced regeneration and additionally highlights incubation temperature as a key parameter for optimizing in vitro tissue culture.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Hormônios/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , TemperaturaRESUMO
Molecular mechanisms of how constant temperatures affect flowering time have been largely characterized in the model plant Arabidopsis thaliana; however, the effect of natural daily variable temperature outside laboratories is only partly explored. Several flowering genes have been shown to play important roles in temperature responses, including PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC), the two genes encoding for the transcription factors (TFs) that act antagonistically to regulate flowering time by activating and repressing floral integrator FLOWERING LOCUS T (FT), respectively. In this study, we have taken a multidisciplinary approach to explore the contribution of PIF4 to the early flowering observed in the daily variable temperature (VAR) and to broaden its transcriptional network using publicly available transcriptomic data. We observed early flowering in the natural accessions Col-0, C24 and their late flowering hybrid C24xCol grown under VAR, as compared with a constant temperature (CON). The loss-of-function mutation of PIF4 exhibits later flowering in VAR in both the Col-0 parent and the C24xCol hybrid, suggesting that PIF4, at least in part, contributes to acceleration of flowering in the VAR condition. To investigate the interplay between PIF4 and its flowering regulator counterparts, FLC and FT, we performed transcriptional analyses and found that VAR increased PIF4 transcription at the end of the day when temperature peaked at 32°C, when FT transcription was also elevated. On the other hand, we observed a decrease in FLC transcription in the 4-week-old plants grown in VAR, as well as in the plants with PIF4 overexpression grown in CON. These results raise a possibility that PIF4 might also regulate FT indirectly through the repression of FLC, in addition to the well-characterized direct control of PIF4 over FT. To further expand our view on the PIF4-orientated flowering gene network in response to temperature changes, we have constructed a coexpression-transcriptional regulatory network by combining publicly available transcriptomic data and gene regulatory interactions of PIF4 and its closely related flowering genes, PIF5, FLC, and ELF3. The network model reveals conserved and tissue-specific regulatory functions, which are useful for confirming as well as predicting the functions and regulatory interactions between these key flowering genes.
RESUMO
Light perception at dawn plays a key role in coordinating multiple molecular processes and in entraining the plant circadian clock. The Arabidopsis mutant lacking the main photoreceptors, however, still shows clock entrainment, indicating that the integration of light into the morning transcriptome is not well understood. In this study, we performed a high-resolution RNA-sequencing time-series experiment, sampling every 2 min beginning at dawn. In parallel experiments, we perturbed temperature, the circadian clock, photoreceptor signaling, and chloroplast-derived light signaling. We used these data to infer a gene network that describes the gene expression dynamics after light stimulus in the morning, and then validated key edges. By sampling time points at high density, we are able to identify three light- and temperature-sensitive bursts of transcription factor activity, one of which lasts for only about 8 min. Phytochrome and cryptochrome mutants cause a delay in the transcriptional bursts at dawn, and completely remove a burst of expression in key photomorphogenesis genes (HY5 and BBX family). Our complete network is available online (http://www-users.york.ac.uk/â¼de656/dawnBurst/dawnBurst.html). Taken together, our results show that phytochrome and cryptochrome signaling is required for fine-tuning the dawn transcriptional response to light, but separate pathways can robustly activate much of the program in their absence.
Assuntos
Arabidopsis/fisiologia , Ritmo Circadiano/fisiologia , Criptocromos/fisiologia , Células Fotorreceptoras , Fitocromo B/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Luz , Transdução de Sinais , Temperatura , Fatores de TranscriçãoRESUMO
In temperate trees, optimal timing and quality of flowering directly depend on adequate winter dormancy progression, regulated by a combination of chilling and warm temperatures. Physiological, genetic and functional genomic studies have shown that hormones play a key role in bud dormancy establishment, maintenance and release. We combined physiological and transcriptional analyses, quantification of abscisic acid (ABA) and gibberellins (GAs), and modeling to further investigate how these signaling pathways are associated with dormancy progression in the flower buds of two sweet cherry cultivars. Our results demonstrated that GA-associated pathways have distinct functions and may be differentially related with dormancy. In addition, ABA levels rise at the onset of dormancy, associated with enhanced expression of ABA biosynthesis PavNCED genes, and decreased prior to dormancy release. Following the observations that ABA levels are correlated with dormancy depth, we identified PavUG71B6, a sweet cherry UDP-GLYCOSYLTRANSFERASE gene that up-regulates active catabolism of ABA to ABA glucosyl ester (ABA-GE) and may be associated with low ABA content in the early cultivar. Subsequently, we modeled ABA content and dormancy behavior in three cultivars based on the expression of a small set of genes regulating ABA levels. These results strongly suggest the central role of ABA pathway in the control of dormancy progression and open up new perspectives for the development of molecular-based phenological modeling.
Assuntos
Prunus avium , Ácido Abscísico , Flores/genética , Regulação da Expressão Gênica de Plantas , Giberelinas , Dormência de PlantasRESUMO
Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Priônicas/química , Temperatura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Aclimatação/fisiologia , Arabidopsis/química , Temperatura Alta , Modelos Moleculares , Peptídeos/metabolismo , Transição de Fase , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/metabolismoRESUMO
Temperature is a major environmental cue affecting plant growth and development. Plants often experience higher temperatures in the context of a 24 h day-night cycle, with temperatures peaking in the middle of the day. Here, we find that the transcript encoding the bHLH transcription factor PIF7 undergoes a direct increase in translation in response to warmer temperature. Diurnal expression of PIF7 transcript gates this response, allowing PIF7 protein to quickly accumulate in response to warm daytime temperature. Enhanced PIF7 protein levels directly activate the thermomorphogenesis pathway by inducing the transcription of key genes such as the auxin biosynthetic gene YUCCA8, and are necessary for thermomorphogenesis to occur under warm cycling daytime temperatures. The temperature-dependent translational enhancement of PIF7 messenger RNA is mediated by the formation of an RNA hairpin within its 5' untranslated region, which adopts an alternative conformation at higher temperature, leading to increased protein synthesis. We identified similar hairpin sequences that control translation in additional transcripts including WRKY22 and the key heat shock regulator HSFA2, suggesting that this is a conserved mechanism enabling plants to respond and adapt rapidly to high temperatures.
Assuntos
Arabidopsis/crescimento & desenvolvimento , RNA de Plantas/fisiologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Ritmo Circadiano , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica de Plantas , Temperatura , Fatores de Transcrição/fisiologiaRESUMO
Upon detecting abiotic or biotic stress, plants generally reduce their growth, enabling resources to be conserved and diverted to stress response mechanisms. In Arabidopsis thaliana, the AT-hook motif nuclear-localized (AHL) transcription factor family has been implicated in restricting rosette growth in response to stress. However, the mechanism by which AHLs repress growth in rosettes is unknown. In this study, we establish that SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) and other AHLs restrict petiole elongation by antagonizing the growth-promoting PHYTOCHROME-INTERACTING FACTORs (PIFs). Our data show that high levels of SOB3 expression lead to a short-petiole phenotype similar to that conferred by removal of PIF4. Conversely, the dominant-negative sob3-6 mutant has long petioles, a phenotype which is PIF-dependent. We further show that AHLs repress the expression of many PIF-activated genes, several of which are involved in hormone-mediated promotion of growth. Additionally, a subset of PIF-activated, AHL-repressed genes are directly bound by both SOB3 and PIFs. Finally, SOB3 reduces binding of PIF4 to shared target loci. Collectively, our results demonstrate that AHLs repress petiole growth by antagonizing PIF-mediated transcriptional activation of genes associated with growth and hormone pathways. By elucidating a mechanism via which the stress-responsive AHL transcription factor family influences growth in petioles, this study identifies a key step in the gene regulatory network controlling leaf growth in response to the environment.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Folhas de Planta/crescimento & desenvolvimento , Ativação Transcricional , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Folhas de Planta/genética , Transdução de SinaisRESUMO
The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.