Transcriptional feedback loops constitute the molecular circuitry of the plant circadian clock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains thus suggesting it cannot directly regulate CCA1. Here, we established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a clock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Significance Cellular signals evoke rapid and broad changes in gene regulatory networks. To uncover these network dynamics, we developed an approach able to monitor primary targets of a transcription factor (TF) based solely on gene regulation, in the absence of detectable binding. This enabled us to follow the transient propagation of a nitrogen (N) nutrient signal as a direct impact of the master TF Basic Leucine Zipper 1 (bZIP1). Unexpectedly, the largest class of primary targets that exhibit transient associations with bZIP1 is uniquely relevant to the rapid and dynamic propagation of the N signal. Our ability to uncover this transient network architecture has revealed the “dark matter” of dynamic N nutrient signaling in plants that has previously eluded detection.

The pseudoresponse regulators (PRRs) participate in the progression of the circadian clock in Arabidopsis thaliana. The founding member of the family, TIMING OF CAB EXPRESSION1 (TOC1), is an essential component of the transcriptional network that constitutes the core mechanism of the circadian oscillator. Recent data suggest a role in circadian regulation for all five members of the PRR family; however, the molecular function of TOC1 or any other PRRs remains unknown. In this work, we present evidence for the involvement of PRR3 in the regulation of TOC1 protein stability. PRR3 was temporally coexpressed with TOC1 under different photoperiods, yet its tissue expression was only partially overlapping with that of TOC1, as PRR3 appeared restricted to the vasculature. Decreased expression of PRR3 resulted in reduced levels of TOC1 protein, while overexpression of PRR3 caused an increase in the levels of TOC1, all without affecting the amount of TOC1 transcript. PRR3 was able to bind to TOC1 in yeast and in plants and to perturb TOC1 interaction with ZEITLUPE (ZTL), which targets TOC1 for proteasome-dependent degradation. Together, our results indicate that PRR3 might function to modulate TOC1 stability by hindering ZTL-dependent TOC1 degradation, suggesting the existence of local regulators of clock activity and adding to the growing importance of posttranslational regulation in the design of circadian timing mechanisms in plants.

Summary The Drosophila antenna contains receptor neurons for mechanical, olfactory, thermal and humidity stimuli. Neurons expressing the ionotropic receptor IR40a have been implicated in the selection of an appropriate humidity range [1, 2], but while previous work indicates that insect hygroreceptors may be made up by a ‘triad’ of neurons (with a dry- a cold- and a humid-air responding cell [3]), IR40a expression included only cold- and dry-air cells. Here, we report the identification of the humid-responding neuron that completes the hygrosensory triad in the Drosophila antenna. This cell type expresses the Ir68a gene, and Ir68a mutation perturbs humidity preference. Next, we follow the projections of Ir68a neurons to the brain, and show that they form a distinct glomerulus in the posterior antennal lobe (PAL). In the PAL, a simple sensory map represents related features of the external environment with adjacent ‘hot’, ‘cold’, ‘dry’, and ‘humid’ glomeruli -an organization that allows for both unique and combinatorial sampling by central relay neurons. Indeed, flies avoided dry heat more robustly than humid heat, and this modulation was abolished by silencing dry-air receptors. Consistently, at least one projection neuron type received direct synaptic input from both temperature and dry air glomeruli. Our results further our understanding of humidity sensing in the Drosophila antenna, uncover a neuronal substrate for early sensory integration of temperature and humidity in the brain, and illustrate the logic of how ethologically relevant combinations of sensory cues can be processed together to produce adaptive behavioral responses

All animals must detect noxious stimuli to initiate protective behavior, but the evolutionary origin of nociceptive systems is not well understood. Here, we show that noxious heat and irritant chemicals elicit robust escape behaviors in the planarian Schmidtea mediterranea, and that the conserved ion channel TRPA1 is required for these responses. TRPA1 mutant flies (Drosophila) are also defective in noxious heat responses. Unexpectedly, we find that either planarian or human TRPA1 can restore noxious heat avoidance to TRPA1 mutant Drosophila, even though neither is directly activated by heat. Instead, our data suggest that TRPA1 activation is mediated by H2O2/Reactive Oxygen Species, early markers of tissue damage rapidly produced as a result of heat exposure. Together, our data reveal a core function for TRPA1 in noxious heat transduction, demonstrate its conservation from planarians to humans, and imply that animal nociceptive systems may share a common ancestry, tracing back to a progenitor that lived more than 500 million years ago.

Highlights d The fly antenna contains 3 types of cold-activated sensory neurons d Cold cells converge on ''cold'' domain of the brain thermosensory map d Second-order TPN-IIs relay absolute cold, below D. melanogaster's favorite 25 C d TPN-IIs inhibit DN1a circadian neurons, adapting sleep to cold/dark conditions

Simple innate behavior is often described as hard-wired and largely inflexible. Here, we show that the avoidance of hot temperature, a simple innate behavior, contains unexpected plasticity in Drosophila. First, we demonstrate that hot receptor neurons of the antenna and their molecular heat sensor, Gr28B.d, are essential for flies to produce escape turns away from heat. High-resolution fly tracking combined with a 3D simulation of the thermal environment shows that, in steep thermal gradients, the direction of escape turns is determined by minute temperature differences between the antennae (0.1°–1 °C). In parallel, live calcium imaging confirms that such small stimuli reliably activate both peripheral thermosensory neurons and central circuits. Next, based on our measurements, we evolve a fly/vehicle model with two symmetrical sensors and motors (a “Braitenberg vehicle”) which closely approximates basic fly thermotaxis. Critical differences between real flies and the hard-wired vehicle reveal that fly heat avoidance involves decision-making, relies on rapid learning, and is robust to new conditions, features generally associated with more complex behavior.

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P 3 production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved. INTRODUCTIONIn eukaryotes, cell migration is essential for many biological processes such as embryonic development and tissue renewal and in humans it is also linked to numerous pathologies including cancer. Cells are set in motion through reorganization of the actin cytoskeleton that creates cytoplasmic protrusions and promotes forward movement. Members of the Rac subfamily of Rho small GTPases are key regulators of cytoskeletal dynamics in virtually all eukaryotes. Activated Rac proteins relay directional signals from the leading edge of migrating cells to downstream effectors such as SCAR/WAVE and WASP proteins that mediate F-actin polymerization and power cell motility (Jaffe and Hall, 2005;Ridley, 2006;Charest and Firtel, 2007;Kolsch et al., 2008;Ladwein and Rottner, 2008). Despite their importance, the upstream signaling mechanisms that mediate Rac activation during cell migration are still unclear.The activity of the small GTPases is regulated through a GDP/GTP exchange mechanism that is catalyzed by the guanine nucleotide exchange factors (GEF), GTPase-activating proteins (GAPs), and guanine nucleotide-dissociation inhibitors (GDIs). GEFs activate GTPases by catalyzing the dissociation of GDP from the protein, thereby allowing the binding of GTP and their switch to an active state. Members of the classical Dbl homology-pleckstrin homology (DH-PH) domain-containing family were thought to be the universal activators of Rho GTPases until the CZH (CDM-zizimin homology) family of unconventional Rho-GEFs was discovered (Côté and Vuori, 2002;Meller et al., 2005;Côté and Vuori, 2007). In the CZH proteins, a CZH2 domain, rather than a DH domain, interacts wi...