Plants are able to sense and remember heat stress.An initial priming heatstress enables plants to accli-mate so that they are able to survive a subsequent higher temperature.The heat shock transcription factors(HSFs)play a crucial role in this process,but the mechanisms by which plants sense heat stress are not well understood.By comprehensively analyzing the binding targets of all the HSFs,we found that HSFs act in a network,with upstream sensory HSFs acting in a transcriptional cascade to activate downstream HSFs and protective proteins.The upstream sensory HSFs are activated by heat at the protein level via a modular prion-like domain(PrD)structure.PrD1 enables HSF sequestration via chaperone binding,allowing release under heat shock.Activated HSFs are recruited into transcriptionally active foci via PrD2,enabling the for-mation of DNA loops between heat-responsive promoters and enhancer motifs,boosting gene expression days after a priming heat stress.The ability of HSFs to respond rapidly to heat via a protein phase-change response is likely a conserved mechanism in eukaryotes.

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.

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.

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.

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/∼de656/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.

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. The adaptability of the plant Arabidopsis thaliana to different temperatures is regulated by the ability of its ELF3 protein to undergo liquid–liquid phase separation, in a manner that is dependent on the protein’s prion-like domain.

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.

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.

Recently, the expansion of genomic sequencing and genome editing are enabling progress directly in crops themselves. This is key since, while it is highly tractable, Arabidopsis thaliana is a model dicot, and so it will always be important to assess how relevant it is to crop plants. Nevertheless, because it is the most well understood plant for studying flowering time, this chapter begins with a survey of the major concepts that have been found in A. thaliania. It then examines what is known for key crops including tomato, wheat and barley. Finally, it discusses key agronomic challenges and areas of promise. The existence of extensive genetic variation in the responsiveness of the flowering pathways to temperature suggests that within a certain range, farmers and breeders may be able to continue selecting lines best adapted to a new climate.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

Cold temperatures are a threat to temperate plants, and Arabidopsis thaliana has acquired an adaptive gene expression network controlled by CBF transcription factors. The CBFs are sufficient to enable plants to survive otherwise lethal subzero temperatures. Constitutive CBF expression causes delayed flowering and stunted growth, and plants have evolved the ability to restrict CBF expression to occur only in the cold. This allows plants to anticipate likely freezing events and selectively deploy cold tolerance. The mechanism by which cold stress is sensed is however unknown. Here we show that protein translation rates in plants are proportional to temperature, and reduced translation rates trigger a rise in intracellular free calcium that activates the CAMTA transcription factors, and these directly activate cold-induced gene expression.

Day length is a key indicator of seasonal information that determines major patterns of behavior in plants and animals. Photoperiodism has been described in plants for about 100 years, but the underlying molecular mechanisms of day length perception and signal transduction in many systems are not well understood. In trees, photoperiod perception plays a major role in growth cessation during the autumn as well as activating the resumption of shoot growth in the spring, both processes controlled by FLOWERING LOCUS T2 (FT2) expression levels and critical for the survival of perennial plants over winter [1-4]. It has been shown that the conserved role of poplar orthologs to Arabidopsis CONSTANS (CO) directly activates FT2 expression [1, 5]. Overexpression of poplar CO is, however, not sufficient to sustain FT2 expression under short days [5], pointing to the presence of an additional short-day-dependent FT2 repression pathway in poplar. We find that night length information is transmitted via the expression level of a poplar clock gene, LATE ELONGATED HYPOCOTYL 2 (LHY2), which controls FT2 expression. Repression of FT2 is a function of the night extension and LHY2 expression level. We show that LHY2 is necessary and sufficient to activate night length repressive signaling. We propose that the photoperiodic control of shoot growth in poplar involves a balance between FT2 activating and repressing pathways. Our results show that poplar relies on night length measurement to determine photoperiodism through interaction between light signaling pathways and the circadian clock.

Summary 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 PHOTOPERIOD DEPENDENT1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in breeding barley and wheat for more northern latitudes (Faure et al., 2012; Turner, Beales, Faure, Dunford, & Laurie, 2005). How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. We show that in Brachypodium distachyon , phytochrome C (phyC) acts as a molecular timer, directly communicating information to the circadian clock protein ELF3. In this way, ELF3 levels integrate night length information. ELF3 is a central regulator of photoperiodism in Brachypodium, and elf3 mutants display a constitutive long day transcriptome. Conversely, conditions that result in higher levels of ELF3 suppress long day responses. We are able to show that these effects are direct, as ELF3 and phyC occur in a common complex, and they associate with the promoters of a number of conserved regulators of photoperiodism, including PPD1 . Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in SD and is necessary for rapid flowering in response to LD. These findings provide a conceptual framework for understanding observations in the photoperiodic responses of key crops, including wheat, barley and rice.

The Evening Complex interacts with the complex responsible for the deposition of the histone variant H2A.Z, creating repressive chromatin domains to repress a cohort of target genes in Arabidopsis. 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.

The majority of plants use C3 photosynthesis, but over 60 independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor, and Setaria italica as well as C3 Brachypodium distachyon. In C4 species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.

36 The majority of plants use C3 photosynthesis, but over sixty independent lineages of 37 angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene 38 expression is compartmented between mesophyll and bundle sheath cells. We performed 39 DNaseI-SEQ to identify genome-wide profiles of transcription factor binding in leaves of 40 the C4 grasses Zea mays, Sorghum bicolor and Setaria italica as well as C3 Brachypodium 41 distachyon. In C4 species, while bundle sheath strands and whole leaves shared similarity 42 in the broad regions of DNA accessible to transcription factors, the short sequences bound 43 varied. Transcription factor binding was prevalent in gene bodies as well as promoters, 44 and many of these sites could represent duons that impact gene regulation in addition to 45 amino acid sequence. Although globally there was little correlation between any individual 46 DNaseI footprint and cell-specific gene expression, within individual species transcription 47 factor binding to the same motifs in multiple genes provided evidence for shared 48 mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific 49 comparisons identified a small number of highly conserved transcription factor binding 50 sites associated with leaves from species that diverged around 60 million years ago. 51 These data therefore provide insight into the architecture associated with C4 52 photosynthesis gene expression in particular and characteristics of transcription factor 53 binding in cereal crops in general. 54 Introduction 55 Most photosynthetic organisms, including crops of global importance such as wheat, 56 rice and potato use the C3 photosynthesis pathway in which Ribulose-Bisphosphate 57 Carboxylase Oxygenase (RuBisCO) catalyses the primary fixation of CO2. However, 58 carboxylation by RuBisCO is competitively inhibited by oxygen binding the active site 59 (Bowes et al., 1971). This oxygenation reaction generates toxic waste-products that are 60 recycled by an energy-demanding series of metabolic reactions known as photorespiration 61 (Bauwe et al., 2010; Tolbert, 1971). The ratio of oxygenation to carboxylation increases 62 with temperature (Jordan and Ogren, 1984; Sharwood et al., 2016) and so losses from 63 photorespiration are particularly high in the tropics. 64 Multiple plant lineages have evolved mechanisms that suppress oxygenation by 65 concentrating CO2 around RuBisCO. One such strategy is known as C4 photosynthesis. 66 Species that use the C4 pathway include maize, sorghum and sugarcane, and they 67 represent the most productive crops on the planet (Sage and Zhu, 2011). In C4 leaves, 68 additional expenditure of ATP, alterations to leaf anatomy and cellular ultrastructure, as 69 well as spatial separation of photosynthesis between compartments (Hatch, 1987) allows 70 CO2 concentration to be increased around tenfold compared with that in the atmosphere 71 (Furbank, 2011). Despite the complexity of C4 photosynthesis, it is found in over 60 72 independent plant lineages (Sage et al., 2011). In most C4 plants the initial RuBisCO73 independent fixation of CO2 and the subsequent RuBisCO-dependent reactions take place 74 in distinct cell-types known as mesophyll and bundle sheath cells. Although the spatial 75 patterning of gene expression that generates these metabolic specialisations is 76 fundamental to C4 photosynthesis very few examples of cis-elements or trans-factors that 77 restrict gene expression to mesophyll or bundle sheath cells of C4 plants have been 78 identified (Brown et al., 2011; Gowik et al., 2004; Williams et al., 2016; Reyna-Llorens et 79 al., 2018). Moreover, in grasses more generally the DNA-binding properties of relatively 80 few transcription factors have been validated (Bolduc and Hake, 2009; Yu et al., 2015; 81 Eveland et al., 2014; Pautler et al., 2015). In summary, in both C3 and C4 species, work 82 has focussed on analysis of mechanisms controlling the expression of individual genes, 83 and so our understanding of the overall landscape associated with photosynthesis gene 84 expression is poor. 85 In yeast and animal systems, the high sensitivity of open chromatin to DNaseI (Zentner 86 and Henikoff, 2014) has allowed comprehensive, genome-wide characterization of 87 transcription factor binding sites at single nucleotide resolution (Hesselberth et al., 2009; 88 Neph et al., 2012; Thurman et al., 2012). In plants, DNaseI-SEQ and more recently Assay 89 for Transposase-Accessible Chromatin (ATAC-SEQ) have been employed in C3 species 90 and provided insight into the patterns of transcription factor binding associated with 91 development (Zhang et al., 2012a; Pajoro et al., 2014; Zhang et al., 2012b, 2016), heat 92 stress (Sullivan et al., 2014) and root cell differentiation (Maher et al., 2017). By carrying 93 out DNaseI-SEQ on grass leaves that use either C3 or C4 photosynthesis, we aimed to 94 provide insight into the transcription factor binding repertoire associated with each form of 95 photosynthesis. Our data indicate more transcription factor binding sites are found in gene 96 bodies than promoters, and up to 25% of the footprints represent ‘duons’ – sequences 97 located in exons that have an impact on both gene regulation as well as the amino acid 98 sequence of the protein they encode. It is also clear that specific cell types from leaf tissue 99 make use of a markedly distinct cis-regulatory code and that despite significant turnover in 100 the cistrome of grasses, a small number of transcription factor motifs are conserved across 101 60 million years of evolution. Comparison of sites bound by transcription factors in both C3 102 and C4 leaves demonstrates that the repeated evolution of C4 photosynthesis is built on 103 both the de novo gain of cis-elements and the exaptation of highly conserved regulatory 104 elements found in the ancestral C3 system. 105 Results 106 A cis-regulatory atlas for grasses 107 To provide insight into the regulatory architecture associated with C3 and C4 108 photosynthesis in cereal crops, four grass were selected. Brachypodium distachyon uses 109 the ancestral C3 pathway (Figure 1A). Sorghum bicolor, Zea mays and Setaria italica all 110 use C4 photosynthesis, they were chosen as phylogenetic reconstructions indicate that S. 111 italica represents an independent evolutionary origin of the C4 pathway (Figure 1A) and 112 comparison of these species can provide insight into parallel and convergent evolution of 113 C4 gene expression. Nuclei from a minimum of duplicate samples of S. italica (C4), S. 114 bicolor (C4), Z. mays (C4) and B. distachyon (C3) leaves were treated with DNaseI 115 (Supplemental Figure 1) and subjected to deep sequencing. A total of 806,663,951 reads 116 could be uniquely mapped to the respective genome sequences of these species 117 (Supplemental Table 1). From all four genomes, 159,396 DNaseI-hypersensitive sites 118 (DHS) of between 150-15,060 base pairs representing broad regulatory regions accessible 119 to transcription factor binding were identified (Figure 1B). Between 20,817 and 27,746 120 genes were annotated as containing at least one DHS (Supplemental Table 2). For 121 subsequent analysis, only DHS that were consistent between replicates as determined by 122 the Irreproducible Discovery Rate framework (Li and Dewey, 2011) were used. 123 DNaseI footprinting is a well-established technique for detecting DNA-protein 124 interactions at base pair resolution and as such has been used to generate Digital 125 Genomic Footprints (DGF) to predict transcription factor binding sites. DGF are obtained 126 by pooling all replicates to maximise the number of reads that map within each DHS, and 127 then modelling differential accumulation of reads mapping to positive or negative strands 128 around transcription factor binding sites within the DHS (Piper et al., 2013). However, the 129 DNaseI enzyme possesses some sequence bias that can affect prediction of transcription 130 factor binding sites (He et al., 2014; Yardimci et al., 2014). After performing DNaseI-SEQ 131 on “naked DNA” that is devoid of nucleosomes from each species, we identified hundreds 132 of DGF that likely represent false positives (Supplemental Figure 2A). For all species, 133 analysis of the DGF derived from naked DNA showed that treatment with DNaseI led to 134 similar sequences being preferentially digested (Supplemental Figure 2B). However, 135 because false positive DGF predicted from this approach will be influenced by the number 136 of reads that map to each genome, and in the case of maize fewer reads mapped in total, 137 the number of false positive DGF varied between species (Supplemental Figure 2A). To 138 overcome this issue, we implemented a more conservative pipeline that rather than 139 defining false positives at specific locations within the genome, calculates DNaseI cutting 140 bias for all hexamers across each genome. By employing a mixture model framework, 141 these data are then used to generate a background signal to estimate footprint likelihood 142 scores for each putative DGF (Yardimici et al., 2014; Supplemental Figure 2B). This 143 approach removed between 15% and 30% of DGF from each sample (Supplemental 144 Figure 2C) and left a total of 430,205 DGF corresponding to individual transcription factor 145 binding sites between 11 and 25 base pairs being identified (Figure 1B&C; Supplemental 146 Table 3). At least one transcription factor footprint was identified in >75% of the broader 147 regions defined by DHS (Supplemental Table 2). 148 We attempted to saturate the number of predicted DGF by sequencing each species at 149 high depth (Supplemental Table 1). In silico sub-sampling of these data indicated that for 150 S. bicolor, S. italica and B. distachyon, the total number of DGF was close to saturation, 151 but for maize despite obtaining 251,955,063 reads from whole leaves this was not the 152 c

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.

Temperature is a key environmental variable influencing plant growth and survival. Protection against high temperature stress in eukaryotes is coordinated by heat shock factors (HSFs), transcription factors that activate the expression of protective chaperones such as HEAT SHOCK PROTEIN 70 (HSP70); however, the pathway by which temperature is sensed and integrated with other environmental signals into adaptive responses is not well understood. Plants are exposed to considerable diurnal variation in temperature, and we have found that there is diurnal variation in thermotolerance in Arabidopsis thaliana, with maximal thermotolerance coinciding with higher HSP70 expression during the day. In a forward genetic screen, we identified a key role for the chloroplast in controlling this response, suggesting that light-induced chloroplast signaling plays a key role. Consistent with this, we are able to globally activate binding of HSFA1a to its targets by altering redox status in planta independently of a heat shock.

The gene regulatory architecture associated with photosynthesis is poorly understood. Most plants use the ancestral C3 pathway, but our most productive cereal crops use C4 photosynthesis. In these C4 cereals, large-scale alterations to gene expression allow photosynthesis to be partitioned between cell types of the leaf. Here we provide a genome-wide transcription factor binding atlas for grasses that operate either C3 or C4 photosynthesis. Most of the >950,000 sites bound by transcription factors are preferentially located in genic sequence rather than promoter regions, and specific families of transcription factors preferentially bind coding sequence. Cell specific patterning of gene expression in C4 leaves is associated with combinatorial modifications to transcription factor binding despite broadly similar patterns of DNA accessibility between cell types. A small number of DNA motifs bound by transcription factors are conserved across 60 million years of grass evolution, and C4 evolution has repeatedly co-opted at least one of these hyper-conserved cis-elements. The grass cistrome is highly divergent from that of the model plant Arabidopsis thaliana.