Sleep is plastic and is influenced by ecological factors and environmental changes. The mechanisms underlying sleep plasticity are not well understood. We show that manipulations that impair flight in Drosophila increase sleep as a form of sleep plasticity. We disrupted flight by blocking the wing-expansion program, genetically disrupting flight, and by mechanical wing perturbations. We defined a new sleep regulatory circuit starting with specific wing sensory neurons, their target projection neurons in the ventral nerve cord, and the neurons they connect to in the central brain. In addition, we identified a critical neuropeptide (burs) and its receptor (rickets) that link wing expansion and sleep. Disrupting flight activates these sleep-promoting projection neurons, as indicated by increased cytosolic calcium levels, and stably increases the number of synapses in their axonal projections. These data reveal an unexpected role for flight in regulating sleep and provide new insight into how sensory processing controls sleep need.

The meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures. We initially assessed the effects of low temperature on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (Triticum aestivum L.) variety ‘Chinese Spring’. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low temperature in a controlled environment room during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after 7 days at 13 °C, suggesting that Ltp1 is deleted in this mutant. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ltp1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. KASP genotyping revealed that the ltp1 mutant has a 4-Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low temperatures is the meiotic recombination gene Dmc1. The ltp1 mutants were subsequently treated at 30 °C for 24 h during meiosis and exhibited a reduced number of crossovers and increased univalence, though to a lesser extent than at 13 °C. We therefore renamed our ltp1 mutant ‘ttmei1’ (temperature-tolerant meiosis 1) to reflect this additional loss of high temperature tolerance.

We have assessed the effects of high and low temperatures on meiotic chromosome synapsis and crossover formation in the hexaploid wheat ( Triticum aestivum L.) variety ‘Chinese Spring’. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene ( Ltp1 ) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low (13°C) or high (30°C) temperatures in controlled environment rooms during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after seven days at 13°C. This suggests that the mutant, which we name ttmei1 ( t emperature t olerance in mei osis 1) has a deletion of a gene that, like Ltp1 , normally stabilises chromosome pairing at low temperatures. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ttmei1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. After 24 hours at 30°C, ttmei1 mutants exhibited a reduced number of crossovers and increased univalence, but to a lesser extent than at 13°C. KASP genotyping revealed that ttmei1 has a 4 Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low (and possibly high) temperatures is the meiotic recombination gene Dmc1 . Key message The meiotic recombination gene Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures.

Sleep is a near universal phenomenon whose function remains controversial. An influential theory of sleep function posits that ecological factors that place animals in harm’s way increase sleep as a state of adaptive inactivity. Here we find that manipulations that impair flight in Drosophila increase sleep. Further, we identify a novel neural pathway from peripheral wing sensory neurons to the central brain that mediates the change in sleep. Moreover, we show that flight impairments activate and induce structural plasticity in specific projection neurons to support increases in sleep over days. Thus, chemosensory neurons do not only signal sensory cues but also appear to provide information on wing-integrity to support behavioural adaptability. Together, these data provide mechanistic support of adaptive increases in sleep and highlight the importance of behavioural flexibility for fitness and survival.

Polyploidization has played an important role in plant evolution. However, upon polyploidization, the process of meiosis must adapt to ensure the proper segregation of increased numbers of chromosomes to produce balanced gametes. It has been suggested that meiotic gene (MG) duplicates return to a single copy following whole genome duplication to stabilize the polyploid genome. Therefore, upon the polyploidization of wheat, a hexaploid species with three related (homeologous) genomes, the stabilization process may have involved rapid changes in content and expression of MGs on homeologous chromosomes (homeologs). To examine this hypothesis, sets of candidate MGs were identified in wheat using co-expression network analysis and orthology informed approaches. In total, 130 RNA-Seq samples from a range of tissues including wheat meiotic anthers were used to define co-expressed modules of genes. Three modules were significantly correlated with meiotic tissue samples but not with other tissue types. These modules were enriched for GO terms related to cell cycle, DNA replication, and chromatin modification and contained orthologs of known MGs. Overall, 74.4% of genes within these meiosis-related modules had three homeologous copies which was similar to other tissue-related modules. Amongst wheat MGs identified by orthology, rather than co-expression, the majority (93.7%) were either retained in hexaploid wheat at the same number of copies (78.4%) or increased in copy number (15.3%) compared to ancestral wheat species. Furthermore, genes within meiosis-related modules showed more balanced expression levels between homeologs than genes in non-meiosis-related modules. Taken together, our results do not support extensive gene loss nor changes in homeolog expression of MGs upon wheat polyploidization. The construction of the MG co-expression network allowed identification of hub genes and provided key targets for future studies.

Wild relatives provide an important source of useful traits in wheat breeding. Wheat and wild relative hybrids have been widely used in breeding programs to introduce such traits into wheat. However, successful introgression is limited by the low frequency of homoeologous crossover (CO) between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70Mb deletion on chromosome 5B ( ph1b ) and wild relatives, have been exploited to increase the level of homoeologous CO, allowing chromosome exchange between their chromosomes. In ph1b -rye hybrids, CO number increases from a mean of 1 CO to 7 COs per cell. CO number can be further increased up to a mean of 12 COs per cell in these ph1b hybrids by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic crossover gene ZIP4 on chromosome 5B ( TaZIP4-B2 ) within the 70Mb deletion, was responsible for the restriction of homoeologous COs in wheat-wild relative hybrids, confirming the ph1b phenotype as a complete Tazip4-B2 deletion mutant ( Tazip4-B2 ph1b) . In this study, we have identified the particular Hoagland solution constituent responsible for the increased chiasma frequency in Tazip4-B2 ph1b mutant-rye hybrids and extended the analysis to Tazip4-B2 TILLING and CRISPR mutant- Ae variabilis hybrids. Chiasma frequency at meiotic metaphase I, in the absence of each Hoagland solution macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O) was analysed. A significant decrease in homoeologous CO frequency was observed when the Mg2+ ion was absent. A significant increase of homoeologous CO frequency was observed in all analysed hybrids, when plants were irrigated with a 1mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat-rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis.

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.

Allopolyploids must possess a mechanism for facilitating synapsis and crossover (CO) between homologues, in preference to homoeologues (related chromosomes), to ensure successful meiosis. In hexaploid wheat, the Ph1 locus has a major effect on the control of these processes. Studying a wheat mutant lacking Ph1 provides an opportunity to explore the underlying mechanisms. Recently, it was proposed that Ph1 stabilises wheat during meiosis, both by promoting homologue synapsis during early meiosis and preventing MLH1 sites on synapsed homoeologues from becoming COs later in meiosis. Here, we explore these two effects and demonstrate firstly that whether or not Ph1 is present, synapsis between homoeologues does not take place during the telomere bouquet stage, with only homologous synapsis taking place during this stage. Furthermore, in wheat lacking Ph1, overall synapsis is delayed with respect to the telomere bouquet, with more synapsis occurring after the bouquet stage, when homoeologous synapsis is also possible. Secondly, we show that in the absence of Ph1, we can increase the number of MLH1 sites progressing to COs by altering environmental growing conditions; we show that higher nutrient levels in the soil or lower temperatures increase the level of both homologue and homoeologue COs. These observations suggest opportunities to improve the exploitation of the Ph1 wheat mutant in breeding programmes.

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently downregulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin decondensation.

Specific labeling of proteins and nucleic acids by immunofluorescence or in situ techniques is an important adjunct to microscopical analysis for cell biology. Labeling of nuclear structures in intact complex tissues is often hampered by problems of penetration of the macromolecular labeling reagents needed. Here we describe a method of labeling isolated plant nuclei that we have found to be a useful approach that can help to overcome these problems.

BACKGROUND:Recent developments in both microscopy and fluorescent protein technologies have made live imaging a powerful tool for the study of plant cells. However, the complications of keeping plant material alive during a long duration experiment while maintaining maximum resolution has limited the use of these methods. RESULTS:Here, we describe an imaging chamber designed to overcome these limitations, which is flexible enough to support a range of sizes of plant materials. We were able use confocal microscopy to follow growth and development of plant cells and tissues over several days. The chamber design is based on a perfusion system, so that the addition of drugs and other experimental treatments are also possible. CONCLUSIONS:In this article we present a design of imaging chamber that makes it possible to image plant material with high resolution for extended periods of time.

Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al., 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.

© Springer Science+Business Media New York 2015 O. Pontes, H. Jin (eds.), Nuclear Functions in Plant Transcription, Signaling and Development, DOI 10.1007/978-1-4939-2386-1_4 A. P. Santos () · M. Oliveira Genomics of Plant Stress Unit, Instituto de Biologia Experimental e Tecnológica, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780–157 Oeiras, Portugal e-mail: apsantos@itqb.unl.pt

The fruitfly, Drosophila melanogaster, has become a critical model system for investigating sleep functions. Most studies use duration of inactivity to measure sleep. However, a defining criterion for sleep is decreased behavioral responsiveness to stimuli. Here we introduce the Drosophila ARousal Tracking system (DART), an integrated platform for efficiently tracking and probing arousal levels in animals. This video-based platform delivers positional and locomotion data, behavioral responsiveness to stimuli, sleep intensity measures, and homeostatic regulation effects - all in one combined system. We show how insight into dynamically changing arousal thresholds is crucial for any sleep study in flies. We first find that arousal probing uncovers different sleep intensity profiles among related genetic background strains previously assumed to have equivalent sleep patterns. We then show how sleep duration and sleep intensity can be uncoupled, with distinct manipulations of dopamine function producing opposite effects on sleep duration but similar sleep intensity defects. We conclude by providing a multi-dimensional assessment of combined arousal and locomotion metrics in the mutant and background strains. Our approach opens the door for deeper insights into mechanisms of sleep regulation and provides a new method for investigating the role of different genetic manipulations in controlling sleep and arousal.

The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels.

During meiosis, homologous chromosomes synapse and recombine at sites marked by the binding of the mismatch repair protein MLH1. In hexaploid wheat, the Ph1 locus has a major effect on whether crossover occurs between homologues or between related homoeologues. Here we report that-in wheat-rye hybrids where homologues are absent-Ph1 affects neither the level of synapsis nor the number of MLH1. Thus in the case of wheat-wild relative hybrids, Ph1 must affect whether MLH1 sites are able to progress to crossover. The observed level of synapsis implies that Ph1 functions to promote homologue pairing rather than suppress homoeologue pairing in wheat. Therefore, Ph1 stabilises polyploidy in wheat by both promoting homologue pairing and preventing MLH1 sites from becoming crossovers on paired homoeologues during meiosis.

Material Supplemental http://genesdev.cshlp.org/content/suppl/2013/09/06/27.17.1845.DC1.html References http://genesdev.cshlp.org/content/27/17/1845.full.html#ref-list-1 This article cites 34 articles, 10 of which can be accessed free at: Open Access Open Access option. Genes & Development Freely available online through the License Commons Creative . http://creativecommons.org/licenses/by-nc/3.0/ License (Attribution-NonCommercial 3.0 Unported), as described at , is available under a Creative Commons Genes & Development This article, published in