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Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.

Integration of environmental signals with endogenous biological processes is essential for organisms to thrive in their natural environment. Being entrained by periodic environmental changes, the circadian clock incorporates external information to coordinate physiological processes, phasing them to the optimal time of the day and year. Here, we present a pivotal role for the clock component GIGANTEA (GI) as a genome-wide regulator of transcriptional networks mediating growth and adaptive processes in plants. We provide mechanistic details on how GI integrates endogenous timing with light signaling pathways through the global modulation of PHYTOCHROME-INTERACTING FACTORs (PIFs). Gating of the activity of these transcriptional regulators by GI directly affects a wide array of output rhythms, including photoperiodic growth. Furthermore, we uncover a role for PIFs in mediating light input to the circadian oscillator and show how their regulation by GI is required to set the pace of the clock in response to light-dark cycles.

For plants adapted to bright light, a decrease in the amount of light received can be detrimental to their growth and survival. Consequently, in response to shade from surrounding vegetation, they initiate a suite of molecular and morphological changes known as the shade avoidance response through which stems and petioles elongate in search for light. Under sunlight--night cycles, the plant's responsiveness to shade varies across the day, being maximal at dusk time. While a role for the circadian clock in this regulation has long been proposed, mechanistic understanding of how it is achieved is incomplete. Here, we show that the clock component GIGANTEA (GI) directly interacts with the transcriptional regulator PHYTOCHROME INTERACTING FACTOR 7 (PIF7), a key player in the response to shade. GI represses PIF7 transcriptional activity and the expression of its target genes in response to shade, thereby fine-tuning the magnitude of the response to limiting light conditions. We find that under light/dark cycles, this function of GI is required to adequately modulate the gating of the response to shade at dusk. Importantly, we also show that this circuit primarily operates in epidermal cells, highlighting the relevance of tissue-specific clock-output connections for the regulation of plant development in resonance with the environment. [ABSTRACT FROM AUTHOR]

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

The Aux/IAA proteins are auxin-sensitive repressors that mediate diverse physiological and developmental processes in plants [1, 2]. There are 29 Aux/IAA genes in Arabidopsis that exhibit unique but partially overlapping patterns of expression [3]. Although some studies have suggested that individual Aux/IAA genes have specialized function, genetic analyses of the family have been limited by the scarcity of loss-of-function phenotypes [4]. Furthermore, with a few exceptions, our knowledge of the factors that regulate Aux/IAA expression is limited [1, 5]. We hypothesize that transcriptional control of Aux/IAA genes plays a central role in the establishment of the auxin-signaling pathways that regulate organogenesis, growth, and environmental response. Here, we describe a screen for transcription factors (TFs) that regulate the Aux/IAA genes. We identify TFs from 38 families, including 26 members of the DREB/CBF family. Several DREB/CBF TFs directly promote transcription of the IAA5 and IAA19 genes in response to abiotic stress. Recessive mutations in these IAA genes result in decreased tolerance to stress conditions, demonstrating a role for auxin in abiotic stress. Our results demonstrate that stress pathways interact with the auxin gene regulatory network (GRN) through transcription of the Aux/IAA genes. We propose that the Aux/IAA genes function as hubs that integrate genetic and environmental information to achieve the appropriate developmental or physiological outcome.

From photosynthetic bacteria to mammals, the circadian clock evolved to track diurnal rhythms and enable organisms to anticipate daily recurring changes such as temperature and light. It orchestrates a broad spectrum of physiology such as the sleep/wake and eating/fasting cycles. While we have made tremendous advances in our understanding of the molecular details of the circadian clock mechanism and how it is synchronized with the environment, we still have rudimentary knowledge regarding its connection to help regulate diurnal physiology. One potential reason is the sheer size of the output network. Diurnal/circadian transcriptomic studies are reporting that around 10% of the expressed genome is rhythmically controlled. Zebrafish is an important model system for the study of the core circadian mechanism in vertebrate. As Zebrafish share more than 70% of its genes with human, it could also be an additional model in addition to rodent for exploring the diurnal/circadian output with potential for translational relevance. Here we performed comparative diurnal/circadian transcriptome analysis with established mouse liver and other tissue datasets. First, by combining liver tissue sampling in a 48h time series, transcription profiling using oligonucleotide arrays and bioinformatics analysis, we profiled rhythmic transcripts and identified 2609 rhythmic genes. The comparative analysis revealed interesting features of the output network regarding number of rhythmic genes, proportion of tissue specific genes and the extent of transcription factor family expression. Undoubtedly, the Zebrafish model system will help identify new vertebrate outputs and their regulators and provides leads for further characterization of the diurnal cis-regulatory network.

The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb-like transcription factor CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is able to initiate and set the phase of clock-controlled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.

To compete for nutrients in diverse soil microenvironments, plants proliferate lateral roots preferentially in nutrient-rich zones. For nitrate, root foraging involves local and systemic signaling; however, little is known about the genes that function in the systemic signaling pathway. By using nitrate enhancer DNA to screen a library of Arabidopsis transcription factors in the yeast one-hybrid system, the transcription factor gene TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) was identified. TCP20, which belongs to an ancient, plant-specific gene family that regulates shoot, flower, and embryo development, was implicated in nitrate signaling by its ability to bind DNA in more than 100 nitrate-regulated genes. Analysis of insertion mutants of TCP20 showed that they had normal primary and lateral root growth on homogenous nitrate media but were impaired in preferential lateral root growth (root foraging) on heterogeneous media in split-root plates. Inhibition of preferential lateral root growth was still evident in the mutants even when ammonium was uniformly present in the media, indicating that the TCP20 response was to nitrate. Comparison of tcp20 mutants with those of nlp7 mutants, which are defective in local control of root growth but not in the root-foraging response, indicated that TCP20 function is independent of and distinct from NLP7 function. Further analysis showed that tcp20 mutants lack systemic control of root growth regardless of the local nitrate concentrations. These results indicate that TCP20 plays a key role in the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots.

In Arabidopsis, the circadian clock allows the plant to coordinate daily external signals with internal processes, conferring enhanced fitness and growth vigor. Although external cues such as temperature can entrain the clock, an important feature of the clock is the ability to maintain a relatively constant period over a range of physiological temperatures; this ability is referred to as "temperature compensation." However, how temperature actually is perceived and integrated into the clock molecular circuitry remains largely unknown. In an effort to identify additional regulators of the circadian clock, including putative components that could modulate the clock response to changes in environmental signals, we identified in a previous large-scale screen a transcription factor that interacts with and regulates the promoter activity of a core clock gene. In this report, we characterized this transcription factor, flowering basic helix-loop-helix 1 (FBH1) that binds in vivo to the promoter of the key clock gene circadian clock-associated 1 (CCA1) and regulates its expression. We found that upon temperature changes, overexpression of FBH1 alters the pace of CCA1 expression by causing a period shortening and thus preventing the clock from buffering against this change in temperature. Furthermore, as is consistent with the current mechanistic model of feedback loops observed in the clock regulatory network, we also determined that CCA1 binds in vivo to the FBH1 promoter and regulates its expression. Together these results establish a role for FBH1 as a transcriptional modulator of warm temperature signals and clock responses in Arabidopsis.

Extensive transcriptional networks play major roles in cellular and organismal functions. Transcript levels are in part determined by the combinatorial and overlapping functions of multiple transcription factors (TFs) bound to gene promoters. Thus, TF-promoter interactions provide the basic molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by an increased complexity of network circuitry due to a significant expansion of TF families. Here, we present the construction of a comprehensive collection of Arabidopsis TFs clones created to provide a versatile resource for uncovering TF biological functions. We leveraged this collection by implementing a high-throughput DNA binding assay and identified direct regulators of a key clock gene (CCA1) that provide molecular links between different signaling modules and the circadian clock. The resources introduced in this work will significantly contribute to a better understanding of the transcriptional regulatory landscape of plant genomes.

Climate change is a global concern for all life on our planet, including humans and plants. Plants' growth and development are significantly affected by abiotic stresses, including adverse temperature, inadequate or excess water availability, nutrient deficiency, and salinity. The circadian clock is a master regulator of numerous developmental and metabolic processes in plants. In an effort to identify new clock-related genes and outputs through bioinformatic analysis, we have revealed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) play a crucial role in regulating a wide range of abiotic stress responses and target ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3), a key transcription factor in the plant hormone Abscisic acid (ABA)-signaling pathway. Specifically, we found that CCA1 and LHY regulate the expression of ABF3 under diel conditions, as well as seed germination under salinity. Conversely, ABF3 controls the expression of core clock genes and orchestrates the circadian period in a stress-responsive manner. ABF3 delivers the stress signal to the central oscillator by binding to the promoter of CCA1 and LHY. Overall, our study uncovers the reciprocal regulation between ABF3 and CCA1/LHY and molecular mechanisms underlying the interaction between the circadian clock and abiotic stress. This finding may aid in developing molecular and genetic solutions for plants to survive and thrive in the face of climate change. [ABSTRACT FROM AUTHOR]

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira ("pirulina" strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a "elper"plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain.