Time's up: Epigenetic clocks in plants.

For over a decade, the animal field has led the way in using DNA methylation measurements to construct epigenetic clocks of aging. These clocks can predict organismal age with a level of accuracy that surpasses any other molecular proxy known to date. Evidence is finally emerging that epigenetic clocks also exist in plants. However, these clocks appear to differ from those in animals in some key aspects, including in their ability to measure time beyond the life span of an individual. Clock-like epigenetic changes can be found in plant circadian rhythms (scale: 24 h), during plant aging (scale: weeks/centuries), and across plant lineage evolution (scale: decades/millennia). Here, we provide a first classification of these different types of epigenetic clocks, highlight their main features, and discuss their biological basis.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

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.

Circadian autonomy and rhythmic precision of the Arabidopsis female reproductive organ.

The plant circadian clock regulates essential biological processes including flowering time or petal movement. However, little is known about how the clock functions in flowers. Here, we identified the circadian components and transcriptional networks contributing to the generation of rhythms in pistils, the female reproductive organ. When detached from the rest of the flower, pistils sustain highly precise rhythms, indicating organ-specific circadian autonomy. Analyses of clock mutants and chromatin immunoprecipitation assays showed distinct expression patterns and specific regulatory functions for clock activators and repressors in pistils. Genetic interaction studies also suggested a hierarchy of the repressing activities that provide robustness and precision to the pistil clock. Globally, the circadian function in pistils primarily governs responses to environmental stimuli and photosynthesis and controls pistil growth and seed weight and production. Understanding the circadian intricacies in reproductive organs may prove useful for optimizing plant reproduction and productivity.

Illuminating the Arabidopsis circadian epigenome: Dynamics of histone acetylation and deacetylation.

The circadian clock generates rhythms in biological processes including plant development and metabolism. Light synchronizes the circadian clock with the day and night cycle and also triggers developmental transitions such as germination, or flowering. The circadian and light signaling pathways are closely interconnected and understanding their mechanisms of action and regulation requires the integration of both pathways in their complexity. Here, we provide a glimpse into how chromatin remodeling lies at the interface of the circadian and light signaling regulation. We focus on histone acetylation/deacetylation and the generation of permissive or repressive states for transcription. Several chromatin remodelers intervene in both pathways, suggesting that interaction with specific transcription factors might specify the proper timing or light-dependent responses. Deciphering the repertoire of chromatin remodelers and their interacting transcription factors will provide a view on the circadian and light-dependent epigenetic landscape amenable for mechanistic studies and timely regulation of transcription in plants.

In Vivo Bioluminescence Analyses of Circadian Rhythms in Arabidopsis thaliana Using a Microplate Luminometer.

Our understanding of the circadian clock function in plants has been markedly assisted by studies with the model species Arabidopsis thaliana. Molecular and genetics approaches have delivered a comprehensive view of the transcriptional regulatory networks underlying the Arabidopsis circadian system. The use of the luciferase as a reporter allowed the precise in vivo determination of circadian periods, phases, and amplitudes of clock promoter activities with unprecedented temporal resolution. An increasing repertoire of fine-tuned luciferases together with additional applications such as translational fusions or bioluminescence molecular complementation assays have considerably expanded our view of circadian protein expression and activity, far beyond transcriptional regulation. Further applications have focused on the in vivo simultaneous examination of rhythms in different parts of the plant. The use of intact versus excised plant organs has also provided a glimpse on both the organ-specific and autonomy of the clocks and the importance of long distance communication for circadian function. This chapter provides a basic protocol for in vivo high-throughput monitoring of circadian rhythms in Arabidopsis seedlings using bioluminescent reporters and a microplate luminometer.

The Arabidopsis Circadian Clock and Metabolic Energy: A Question of Time.

A fundamental principle shared by all organisms is the metabolic conversion of nutrients into energy for cellular processes and structural building blocks. A highly precise spatiotemporal programming is required to couple metabolic capacity with energy allocation. Cellular metabolism is also able to adapt to the external time, and the mechanisms governing such an adaptation rely on the circadian clock. Virtually all photosensitive organisms have evolved a self-sustained timekeeping mechanism or circadian clock that anticipates and responds to the 24-h environmental changes that occur during the day and night cycle. This endogenous timing mechanism works in resonance with the environment to control growth, development, responses to stress, and also metabolism. Here, we briefly describe the prevalent role for the circadian clock controlling the timing of mitochondrial activity and cellular energy in Arabidopsis thaliana. Evidence that metabolic signals can in turn feedback to the clock place the spotlight onto the molecular mechanisms and components linking the circadian function with metabolic homeostasis and energy.

Circadian Control of Metabolism by the Clock Component TOC1.

Photosynthesis in chloroplasts during the day and mitochondrial respiration during the night execute nearly opposing reactions that are coordinated with the internal cellular status and the external conditions. Here, we describe a mechanism by which the Arabidopsis clock component TIMING OF CAB EXPRESSION1 (TOC1) contributes to the diurnal regulation of metabolism. Proper expression of TOC1 is important for sustaining cellular energy and for the diel and circadian oscillations of sugars, amino acids and tricarboxylic acid (TCA) cycle intermediates. TOC1 binds to the promoter of the TCA-related gene FUMARASE 2 to repress its expression at night, which results in decreased fumarate accumulation in TOC1 over-expressing plants and increased in toc1-2 mutant. Genetic interaction studies confirmed that over-expression of FUMARASE 2 in TOC1 over-expressing plants alleviates the molecular and physiological energy-deprivation phenotypes of TOC1 over-expressing plants. Thus, we propose that the tandem TOC1-FUMARASE 2 is one of the mechanisms that contribute to the regulation of plant metabolism during the day and night.

Chromatin Dynamics and Transcriptional Control of Circadian Rhythms in Arabidopsis.

Circadian rhythms pervade nearly all aspects of plant growth, physiology, and development. Generation of the rhythms relies on an endogenous timing system or circadian clock that generates 24-hour oscillations in multiple rhythmic outputs. At its bases, the plant circadian function relies on dynamic interactive networks of clock components that regulate each other to generate rhythms at specific phases during the day and night. From the initial discovery more than 13 years ago of a parallelism between the oscillations in chromatin status and the transcriptional rhythms of an Arabidopsis clock gene, a number of studies have later expanded considerably our view on the circadian epigenome and transcriptome landscapes. Here, we describe the most recent identification of chromatin-related factors that are able to directly interact with Arabidopsis clock proteins to shape the transcriptional waveforms of circadian gene expression and clock outputs. We discuss how changes in chromatin marks associate with transcript initiation, elongation, and the rhythms of nascent RNAs, and speculate on future interesting research directions in the field.

A mobile ELF4 delivers circadian temperature information from shoots to roots.

The circadian clock is synchronized by environmental cues, mostly by light and temperature. Explaining how the plant circadian clock responds to temperature oscillations is crucial to understanding plant responsiveness to the environment. Here, we found a prevalent temperature-dependent function of the Arabidopsis clock component EARLY FLOWERING 4 (ELF4) in the root clock. Although the clocks in roots are able to run in the absence of shoots, micrografting assays and mathematical analyses show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information, but trafficking is essential for controlling the period of the root clock in a temperature-dependent manner. Low temperatures favour ELF4 mobility, resulting in a slow-paced root clock, whereas high temperatures decrease movement, leading to a faster clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.

The Evening Complex Establishes Repressive Chromatin Domains Via H2A.Z Deposition.

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.

Diurnal regulation of SDG2 and JMJ14 by circadian clock oscillators orchestrates histone modification rhythms in Arabidopsis.

BACKGROUND: Circadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood. RESULTS: Here we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20-30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression. CONCLUSIONS: Circadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development.

The EC-HDA9 complex rhythmically regulates histone acetylation at the TOC1 promoter in Arabidopsis.

Circadian clocks are conserved time-keeper mechanisms in some prokaryotes and higher eukaryotes. Chromatin modification is emerging as key regulatory mechanism for refining core clock gene expression. Rhythmic changes in histone marks are closely associated to the TIMING OF CAB EXPRESSION 1 (TOC1) Arabidopsis clock gene. However, the chromatin-related modifiers responsible for these marks remain largely unknown. Here, we uncover that the chromatin modifier HISTONE DEACETYLASE 9 (HDA9) and the Evening complex (EC) component EARLY FLOWERING 3 (ELF3) directly interact to regulate the declining phase of TOC1 after its peak expression. We found that HDA9 specifically binds to the TOC1 promoter through the interaction with ELF3. The EC-HDA9 complex promotes H3 deacetylation at the TOC1 locus, contributing to suppressing TOC1 expression during the night, the time of EC function. Therefore, we have identified the mechanism by which the circadian clock intertwines with chromatin-related components to shape the circadian waveforms of gene expression in Arabidopsis.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

Interactive roles of chromatin regulation and circadian clock function in plants.

Circadian rhythms in transcription ultimately result in oscillations of key biological processes. Understanding how transcriptional rhythms are generated in plants provides an opportunity for fine-tuning growth, development, and responses to the environment. Here, we present a succinct description of the plant circadian clock, briefly reviewing a number of recent studies but mostly emphasizing the components and mechanisms connecting chromatin remodeling with transcriptional regulation by the clock. The possibility that intergenomic interactions govern hybrid vigor through epigenetic changes at clock loci and the function of epialleles controlling clock output traits during crop domestication are also discussed.

A Functional Connection between the Circadian Clock and Hormonal Timing in Arabidopsis.

The rotation of the Earth entails changes in environmental conditions that pervasively influence an organism's physiology and metabolism. An internal cellular mechanism known as the circadian clock acts as an internal timekeeper that is able to perceive the changes in environmental cues to generate 24-h rhythms in synchronization with daily and seasonal fluctuations. In plants, the circadian clock function is particularly important and regulates nearly every aspect of plant growth and development as well as proper responses to stresses. The circadian clock does not function in isolation but rather interconnects with an intricate network of different pathways, including those of phytohormones. Here, we describe the interplay of the circadian clock with a subset of hormones in Arabidopsis. The molecular components directly connecting the circadian and hormone pathways are described, highlighting the biological significance of such connections in the control of growth, development, fitness, and survival. We focus on the overlapping as well as contrasting circadian and hormonal functions that together provide a glimpse on how the Arabidopsis circadian system regulates hormone function in response to endogenous and exogenous cues. Examples of feedback regulation from hormone signaling to the clock are also discussed.

Targeted Recruitment of the Basal Transcriptional Machinery by LNK Clock Components Controls the Circadian Rhythms of Nascent RNAs in Arabidopsis.

The rhythms of steady-state mRNA expression pervade nearly all circadian systems. However, the mechanisms behind the rhythmic transcriptional synthesis and its correlation with circadian expression remain fully unexplored, particularly in plants. Here, we discovered a multifunctional protein complex that orchestrates the rhythms of transcriptional activity in Arabidopsis thaliana The expression of the circadian oscillator genes TIMING OF CAB EXPRESSION1/PSEUDO-RESPONSE REGULATOR1 and PSEUDO-RESPONSE REGULATOR5 initially relies on the modular function of the clock-related factor REVEILLE8: its MYB domain provides the DNA binding specificity, while its LCL domain recruits the clock components, NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED proteins (LNKs), to target promoters. LNKs, in turn, specifically interact with RNA Polymerase II and the transcript elongation FACT complex to rhythmically co-occupy the target loci. The functional interaction of these components is central for chromatin status, transcript initiation, and elongation as well as for proper rhythms in nascent RNAs. Thus, our findings explain how genome readout of environmental information ultimately results in rhythmic changes of gene expression.

The Circadian Clock Sets the Time of DNA Replication Licensing to Regulate Growth in Arabidopsis.

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.

The Elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis.

The Elongator complex (hereafter Elongator) promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response and sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, including downregulation of circadian clock components, positive regulators of skoto- or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and by double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling.This article has an associated First Person interview with the first author of the paper.

Identification and characterization of key circadian clock genes of tobacco hairy roots: putative regulatory role in xenobiotic metabolism.

The circadian clock is an endogenous system that allows organisms to daily adapt and optimize their physiology and metabolism. We studied the key circadian clock gene (CCG) orthologs in Nicotiana tabacum seedlings and in hairy root cultures (HRC). Putative genes involved in the metabolism of xenobiotic compounds (MXC) were selected and their expression profiles were also analyzed. Seedlings and HRC displayed similar diurnal variations in the expression profiles for the CCG examined under control conditions (CC). MXC-related genes also showed daily fluctuations with specific peaks of expression. However, when HRC were under phenol treatment (PT), the expression patterns of the clock and MXC-related genes were significantly affected. In 2-week-old HRC, PT downregulated the expression of NtLHY, NtTOC1, and NtPRR9 while NtFKF1 and NtGI genes were upregulated by phenol. In 3-week-old HRC, PT also downregulated the expression of all CCG analyzed and NtTOC1 was the most affected. Following PT, the expression of the MXC-related genes was upregulated or displayed an anti-phasic expression profile compared to the expression under CC. Our studies thus provide a glimpse of the circadian expression of clock genes in tobacco and the use of HRC as a convenient system to study plant responses to xenobiotic stresses.

MYB96 shapes the circadian gating of ABA signaling in Arabidopsis.

Circadian clocks regulate the rhythms of biological activities with a period of approximately 24-hours and synchronize plant metabolism and physiology with the environmental cycles. The clock also gates responses to environmental stresses to maximize fitness advantages. Here we report that the MYB96 transcription factor is connected with the clock oscillator to shape the circadian gating of abscisic acid (ABA) responses. MYB96 directly binds to the TIMING OF CAB EXPRESSION 1 (TOC1) promoter to positively regulate its expression. The use of myb96 mutant plants shows that this regulation is essential for the gated induction of TOC1 by ABA. In turn, MYB96 induction by ABA is also altered in toc1-3 mutant plants. The increased tolerance to drought of MYB96 over-expressing plants is decreased in the toc1-3 mutant background, suggesting that MYB96 and TOC1 intersect the circadian clock and ABA signaling. The MYB96-TOC1 function might be also regulated by the clock component CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), which binds to the MYB96 promoter and alters its circadian expression. Thus, a complex circuitry of CCA1-MYB96-TOC1 regulatory interactions provides the mechanistic basis underlying the connection between circadian and stress signaling to optimize plant fitness to ambient stresses.