ABSTRACT
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.
Subject(s)
Carbon Dioxide , Climate Change , Stress, Physiological , Carbon Dioxide/metabolism , Plant Transpiration/physiology , Plants/metabolism , Water/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Prion Proteins/chemistry , Temperature , Transcription Factors/chemistry , Transcription Factors/metabolism , Acclimatization/physiology , Arabidopsis/chemistry , Hot Temperature , Models, Molecular , Peptides/metabolism , Phase Transition , Protein Domains , Repressor Proteins/chemistry , Repressor Proteins/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Transcription Factors , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Prions , Temperature , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Plants are highly sensitive to temperature and can perceive a difference of as little as 1 degrees C. How temperature is sensed and integrated in development is unknown. In a forward genetic screen in Arabidopsis, we have found that nucleosomes containing the alternative histone H2A.Z are essential to perceiving ambient temperature correctly. Genotypes deficient in incorporating H2A.Z into nucleosomes phenocopy warm grown plants, and show a striking constitutive warm temperature transcriptome. We show that nucleosomes containing H2A.Z display distinct responses to temperature in vivo, independently of transcription. Using purified nucleosomes, we are able to show that H2A.Z confers distinct DNA-unwrapping properties on nucleosomes, indicating a direct mechanism for the perception of temperature through DNA-nucleosome fluctuations. Our results show that H2A.Z-containing nucleosomes provide thermosensory information that is used to coordinate the ambient temperature transcriptome. We observe the same effect in budding yeast, indicating that this is an evolutionarily conserved mechanism.
Subject(s)
Arabidopsis/physiology , Histones/physiology , Nucleosomes/metabolism , Arabidopsis Proteins/metabolism , DNA, Plant/metabolism , Gene Expression Profiling , HSP70 Heat-Shock Proteins/metabolism , Microfilament Proteins/metabolism , TemperatureABSTRACT
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.
Subject(s)
Flowers , Oryza , Photoperiod , Arabidopsis/genetics , Arabidopsis/growth & development , Flowers/genetics , Flowers/growth & development , Flowers/radiation effects , Gene Expression Regulation, Plant , Light , Oryza/genetics , Oryza/growth & development , Oryza/radiation effects , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/metabolism , Circadian Rhythm , DNA, Plant/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant , Arabidopsis/genetics , Arabidopsis Proteins/genetics , DNA, Plant/genetics , DNA-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Histones/metabolism , Hormones/metabolism , Plant Shoots/genetics , Plant Shoots/metabolism , TemperatureABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Phytochrome , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation, Plant , Phytochrome/genetics , Phytochrome/metabolism , Plants/metabolismABSTRACT
The Evening Complex (EC) is a core component of the Arabidopsis (Arabidopsis thaliana) circadian clock, which represses target gene expression at the end of the day and integrates temperature information to coordinate environmental and endogenous signals. Here we show that the EC induces repressive chromatin structure to regulate the evening transcriptome. The EC component ELF3 directly interacts with a protein from the SWI2/SNF2-RELATED (SWR1) complex to control deposition of H2A.Z-nucleosomes at the EC target genes. SWR1 components display circadian oscillation in gene expression with a peak at dusk. In turn, SWR1 is required for the circadian clockwork, as defects in SWR1 activity alter morning-expressed genes. The EC-SWR1 complex binds to the loci of the core clock genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and catalyzes deposition of nucleosomes containing the histone variant H2A.Z coincident with the repression of these genes at dusk. This provides a mechanism by which the circadian clock temporally establishes repressive chromatin domains to shape oscillatory gene expression around dusk.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chromatin/metabolism , Histones/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Circadian Clocks/physiology , Histones/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: Bud dormancy is a crucial stage in perennial trees and allows survival over winter to ensure optimal flowering and fruit production. Recent work highlighted physiological and molecular events occurring during bud dormancy in trees. However, they usually examined bud development or bud dormancy in isolation. In this work, we aimed to further explore the global transcriptional changes happening throughout bud development and dormancy onset, progression and release. RESULTS: Using next-generation sequencing and modelling, we conducted an in-depth transcriptomic analysis for all stages of flower buds in several sweet cherry (Prunus avium L.) cultivars that are characterized for their contrasted dates of dormancy release. We find that buds in organogenesis, paradormancy, endodormancy and ecodormancy stages are defined by the expression of genes involved in specific pathways, and these are conserved between different sweet cherry cultivars. In particular, we found that DORMANCY ASSOCIATED MADS-box (DAM), floral identity and organogenesis genes are up-regulated during the pre-dormancy stages while endodormancy is characterized by a complex array of signalling pathways, including cold response genes, ABA and oxidation-reduction processes. After dormancy release, genes associated with global cell activity, division and differentiation are activated during ecodormancy and growth resumption. We then went a step beyond the global transcriptomic analysis and we developed a model based on the transcriptional profiles of just seven genes to accurately predict the main bud dormancy stages. CONCLUSIONS: Overall, this study has allowed us to better understand the transcriptional changes occurring throughout the different phases of flower bud development, from bud formation in the summer to flowering in the following spring. Our work sets the stage for the development of fast and cost effective diagnostic tools to molecularly define the dormancy stages. Such integrative approaches will therefore be extremely useful for a better comprehension of complex phenological processes in many species.
Subject(s)
Gene Expression Profiling/methods , Plant Dormancy , Plant Proteins/genetics , Prunus avium/physiology , Flowers/genetics , Flowers/physiology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , High-Throughput Nucleotide Sequencing , Models, Genetic , Prunus avium/geneticsABSTRACT
Plants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes; these expansions are linked to adaptation to environmental stressors. Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determined that the flanking sequences near G-boxes help determine in vitro specificity but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we constructed a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a Web site that provides interactive visualizations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations.
Subject(s)
Arabidopsis/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/genetics , G-Box Binding Factors/genetics , Gene Expression Regulation, Plant/genetics , Gene Regulatory Networks , Nucleotide Motifs , Plant Proteins/geneticsABSTRACT
Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Flowers/growth & development , Flowers/metabolism , Temperature , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Plant , Photoperiod , Plant Leaves/metabolism , Promoter Regions, Genetic/genetics , Signal Transduction , Time FactorsABSTRACT
A key challenge for understanding transcriptional regulation is being able to measure transcription factor (TF)-DNA binding events with sufficient spatial and temporal resolution; that is, when and where TFs occupy their cognate sites. A recent study by Para et al. has highlighted the dynamics underlying the activation of gene expression by a master regulator TF. This study provides concrete evidence for a long-standing hypothesis in biology, the "hit-and-run" mechanism, which was first proposed decades ago. That is, gene expression is dynamically controlled by a TF that transiently binds and activates a target gene, which might stay in a transcriptionally active state after the initial binding event has ended. Importantly, the experimental procedure introduced, TARGET, provides a useful way for identifying multiple target genes transiently bound by their regulators, which can be used in conjunction with other well-established methods to improve our understanding of transcriptional regulatory dynamics.
Subject(s)
Transcription Factors/physiology , Animals , Base Sequence , Binding Sites , Gene Expression Regulation , Gene Regulatory Networks , Humans , Protein Binding , Regulatory Sequences, Nucleic AcidABSTRACT
During flowering, primordia on the flanks of the shoot apical meristem are specified to form flowers instead of leaves. Like many plants, Arabidopsis thaliana integrates environmental and endogenous signals to control the timing of reproduction. To study the underlying regulatory logic of the floral transition, we used a combination of modeling and experiments to define a core gene regulatory network. We show that FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) act through FD and FD PARALOG to regulate the transition. The major floral meristem identity gene LEAFY (LFY) directly activates FD, creating a positive feedback loop. This network predicts flowering behavior for different genotypes and displays key properties of the floral transition, such as signal integration and irreversibility. Furthermore, modeling suggests that the control of TFL1 is important to flexibly counterbalance incoming FT signals, allowing a pool of undifferentiated cells to be maintained despite strong differentiation signals in nearby cells. This regulatory system requires TFL1 expression to rise in proportion to the strength of the floral inductive signal. In this network, low initial levels of LFY or TFL1 expression are sufficient to tip the system into either a stable flowering or vegetative state upon floral induction.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Flowers/physiology , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cell Differentiation , Feedback, Physiological , Flowers/genetics , Flowers/metabolism , Gene Expression Regulation, Plant , Gene Regulatory Networks , Genes, Plant , Genotype , Meristem/genetics , Meristem/metabolism , Models, Molecular , Plant Cells/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The freshwater cnidarian Hydra was first described in 1702 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals. Today, Hydra is an important model for studies of axial patterning, stem cell biology and regeneration. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann-Mangold organizer, pluripotency genes and the neuromuscular junction.
Subject(s)
Genome/genetics , Hydra/genetics , Animals , Anthozoa/genetics , Comamonadaceae/genetics , DNA Transposable Elements/genetics , Gene Transfer, Horizontal/genetics , Genome, Bacterial/genetics , Hydra/microbiology , Hydra/ultrastructure , Molecular Sequence Data , Neuromuscular Junction/ultrastructureABSTRACT
At high ambient temperature, plants display dramatic stem elongation in an adaptive response to heat. This response is mediated by elevated levels of the phytohormone auxin and requires auxin biosynthesis, signaling, and transport pathways. The mechanisms by which higher temperature results in greater auxin accumulation are unknown, however. A basic helix-loop-helix transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), is also required for hypocotyl elongation in response to high temperature. PIF4 also acts redundantly with its homolog, PIF5, to regulate diurnal growth rhythms and elongation responses to the threat of vegetative shade. PIF4 activity is reportedly limited in part by binding to both the basic helix-loop-helix protein LONG HYPOCOTYL IN FAR RED 1 and the DELLA family of growth-repressing proteins. Despite the importance of PIF4 in integrating multiple environmental signals, the mechanisms by which PIF4 controls growth are unknown. Here we demonstrate that PIF4 regulates levels of auxin and the expression of key auxin biosynthesis genes at high temperature. We also identify a family of SMALL AUXIN UP RNA (SAUR) genes that are expressed at high temperature in a PIF4-dependent manner and promote elongation growth. Taken together, our results demonstrate direct molecular links among PIF4, auxin, and elongation growth at high temperature.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Hot Temperature , Indoleacetic Acids/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Plant , Genes, Plant/genetics , Promoter Regions, Genetic/genetics , Protein BindingABSTRACT
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.
Subject(s)
DNA-Binding Proteins , DNA , Electrophoretic Mobility Shift Assay , Temperature , DNA-Binding Proteins/metabolism , Protein Binding , DNA/chemistry , Electrophoresis, Polyacrylamide GelABSTRACT
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.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Circadian Clocks , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Transcription Factors/metabolism , Phase Separation , Mutation , Light , Circadian Clocks/physiology , Gene Expression Regulation, Plant , Circadian Rhythm/physiologyABSTRACT
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.
Subject(s)
Fluorescence Resonance Energy Transfer , RNA , RNA/chemistry , Temperature , Circular Dichroism , OligonucleotidesABSTRACT
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.