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Gene structural annotation is a critical step in obtaining biological knowledge from genome sequences yet remains a major challenge in genomics projects. Currentde novoHidden Markov Models are limited in their capacity to model biological complexity; while current pipelines are resource-intensive and their results vary in quality with the available extrinsic data. Here, we build on our previous work in applying Deep Learning to gene calling to make a fully applicable, fast and user friendly tool for predicting primary gene models from DNA sequence alone. The quality is state-of-the-art, with predictions scoring closer by most measures to the references than to predictions from otherde novotools. Helixer’s predictions can be used as is or could be integrated in pipelines to boost quality further. Moreover, there is substantial potential for further improvements and advancements in gene calling with Deep Learning.Helixer is open source and available athttps://github.com/weberlab-hhu/HelixerA web interface is available athttps://www.plabipd.de/helixer_main.html

Plant omics research has advanced to the stage where it is feasible to generate data from multiple samples and multiple time points to gain insight into biological processes. This impressive array of data can prove challenging to interpret. In this chapter, we describe a solution to this problem, consisting of the MapMan transcript visualization application and the associated MapMan4 ontology and Mercator4 online annotation process.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet., Current Biology, 30 (12), ISSN:0960-9822, ISSN:1879-0445

Highlight Expression of cell wall-related genes marks the onset of haustorium development in the parasitic plant Cuscuta. Action assays suggest a central role for xyloglucan endotransglucosylases/hydrolases in host plant infection., Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium.

Membrane proteins are estimated to constitute a quarter of all proteins encoded in plant genomes, yet only a limited number have been experimentally characterized. This is mainly due to the large variation in particular physical properties coupled with purification difficulties. Computational methods are therefore very helpful for the initial characterization of a candidate membrane protein. Individual prediction tools can, with varying levels of success, predict the occurrence of transmembrane spans, the subcellular location, and lipid posttranslational modifications. Since it can be tedious to consult each prediction tool separately, ARAMEMNON has been designed to compile various computational predictions for plant membrane proteins and to present the results via a user-friendly web interface. This protocol describes how to use ARAMEMNON to identify and characterize plant membrane proteins.

Membrane proteins are estimated to constitute a quarter of all proteins encoded in plant genomes, yet only a limited number have been experimentally characterized. This is mainly due to the large variation in particular physical properties coupled with purification difficulties. Computational methods are therefore very helpful for the initial characterization of a candidate membrane protein. Individual prediction tools can, with varying levels of success, predict the occurrence of transmembrane spans, the subcellular location, and lipid posttranslational modifications. Since it can be tedious to consult each prediction tool separately, ARAMEMNON has been designed to compile various computational predictions for plant membrane proteins and to present the results via a user-friendly web interface. This protocol describes how to use ARAMEMNON to identify and characterize plant membrane proteins.

Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

Induction and mobilization of transposable elements (TEs) following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associated with long non-coding RNAs (lncRNAs), many of which are also upregulated by stress. Yet, it is unknown whether DNA damage-induced transcriptional activation of TEs and lncRNAs occurs sporadically or is a synchronized, genome-wide response. Here we investigated the transcriptome of Arabidopsis wild-type (WT) and ataxia telangiectasia mutated (atm) mutant plants 3 h after induction of DNA damage. In WT, expression of 5.2% of the protein-coding genes is ≥2-fold changed, whereas in atm plants, only 2.6% of these genes are regulated, and the response of genes associated with DNA repair, replication, and cell cycle is largely lost. In contrast, only less than 0.6% of TEs and lncRNAs respond to DNA damage in WT plants, and the regulation of ≥95% of them is ATM-dependent. The ATM-downstream factors BRCA1, DRM1, JMJ30, AGO2, and the ATM-independent AGO4 participate in the regulation of individual TEs and lncRNAs. Remarkably, protein-coding genes located adjacent to DNA damage-responsive TEs and lncRNAs are frequently coexpressed, which is consistent with the hypothesis that TEs and lncRNAs located close to genes commonly function as controlling elements.

Plastid Origins The glaucophytes, represented by the alga Cyanophora paradoxa , are the putative sister group of red and green algae and plants, which together comprise the founding group of photosynthetic eukaryotes, the Plantae. In their analysis of the genome of C. paradoxa , Price et al. (p. 843 ; see the Perspective by Spiegel ) demonstrate a unique origin for the plastid in the ancestor of this supergroup, which retains much of the ancestral diversity in genes involved in carbohydrate metabolism and fermentation, as well as in the gene content of the mitochondrial genome. Moreover, about 3.3% of nuclear genes in C. paradoxa seem to carry a signal of cyanobacterial ancestry, and key genes involved in starch biosynthesis are derived from energy parasites such as Chlamydiae. Rapid radiation, reticulate evolution via horizontal gene transfer, high rates of gene divergence, loss, and replacement, may have diffused the evolutionary signals within this supergroup, which perhaps explains previous difficulties in resolving its evolutionary history.

A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network.

In all living organisms transmembrane (TM) proteins are crucially involved in many physiological processes and constitute 20-30% of the proteome. An important class of TM proteins are transporters that interconnect biochemical pathways across the plasma membrane and intracellular membranes, e.g. the mitochondrial membranes and chloroplast envelope membranes. In recent years, bioinformatical tools to predict TM domains and subcellular localization were developed and used to analyze the first complete plant genomes of Arabidopsis and rice. This review focuses on plant TM proteome databases that compile topology and intracellular targeting predictions and different kinds of experimental data. In addition, other web sites are discussed that contribute useful experimental and/or bioinformatical data.

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de.

The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)-specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6-phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6-bisphosphatase. Despite its regulatory role in the feed-forward control of sucrose biosynthesis, variations in the fructose 2,6-bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt-1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1-1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt-1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady-state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.

Regulation of uptake and compartmentation of metal ions is important for the maintenance of metal ion homeostasis. To identify mechanisms involved in the protection of plants from Mn toxicity, wild-type yeast was transformed with an Arabidopsis cDNA library and transformants were screened on toxic Mn concentrations. Wild-type yeast could not grow in the presence of 30 mM MnSO4, while two transformants carrying variants of the same gene were able to grow. Database searches revealed that the isolated cDNAs correspond to AtCAX2, previously described as a vacuolar calcium-proton antiporter. Since no other genes could be identified, AtCAX2 might represent a major function permitting Mn detoxification in this suppressor screen. Furthermore, yeast transformed with the two AtCAX2 cDNAs showed increased sensitivity towards hydrogen peroxide, pointing to a limited availability of cytoplasmic Mn in the presence of AtCAX2 activity. The open reading frames of the cDNA encoded polypeptides that have a 42 and a 92 amino acids shorter N-terminal region relative to the predicted full-length coding region of AtCAX2. In contrast to both truncated cDNAs, the full-length clone was unable to confer Mn resistance to yeast, indicating that, similar to AtCAX1, AtCAX2 also carries an autoinhibitory N-terminal domain regulating the activity of AtCAX2.

Björn Usadel and colleagues report the genome sequence of the wild tomato species Solanum pennellii. The authors identify genes important for stress tolerance, metabolism and fruit maturation and suggest that transposable elements have had an important role in the evolution of the S. penellii stress response. Supplementary information The online version of this article (doi:10.1038/ng.3046) contains supplementary material, which is available to authorized users., Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum1. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance2. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm. Supplementary information The online version of this article (doi:10.1038/ng.3046) contains supplementary material, which is available to authorized users.

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

A substantial part of the Earths’ soil organic carbon (SOC) is stored in Arctic permafrost peatlands, which represent large potential sources for increased emissions of the greenhouse gases CH4 and CO2 in a warming climate. The microbial communities and their genetic repertoire involved in the breakdown and mineralisation of SOC in these soils are, however, poorly understood. In this study, we applied a combined metagenomic and metatranscriptomic approach on two Arctic peat soils to investigate the identity and the gene pool of the microbiota driving the SOC degradation in the seasonally thawed active layers. A large and diverse set of genes encoding plant polymer-degrading enzymes was found, comparable to microbiotas from temperate and subtropical soils. This indicates that the metabolic potential for SOC degradation in Arctic peat is not different from that of other climatic zones. The majority of these genes were assigned to three bacterial phyla, Actinobacteria, Verrucomicrobia and Bacteroidetes. Anaerobic metabolic pathways and the fraction of methanogenic archaea increased with peat depth, evident for a gradual transition from aerobic to anaerobic lifestyles. A population of CH4-oxidising bacteria closely related to Methylobacter tundripaludum was the dominating active group of methanotrophs. Based on the in-depth characterisation of the microbes and their genes, we conclude that these Arctic peat soils will turn into CO2 sources owing to increased active layer depth and prolonged growing season. However, the extent of future CH4 emissions will critically depend on the response of the methanotrophic bacteria. The ISME Journal (2013) 7, 299–311; doi:10.1038/ismej.2012.99; published online 6 September 2012 Subject Category: integrated genomics and post-genomics approaches in microbial ecology

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387) pPPIs between 179 proteins, yielding a scale-free network (r2=0.863). Eighty of 142 transmembrane receptor-like kinases (RLK) tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naive Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

Plastids semi-autonomous, membrane-bound organelles of plant cells that carry out a large number of biosynthetic and other functions such as photosynthesis (chloroplasts), ammonia and sulfur assimilation, and starch storage (amyloplasts) are metabolically extraordinarily active and versatile organelles that are found in all plant cells with the exception of angiosperm pollen grains. Many of the plastid-localized biochemical pathways depend on precursors from the cytosol and, in turn, many cytosolic pathways depend on the supply of precursor molecules from the plastid stroma. Hence, a massive traffic of metabolites occurs across the permeability barrier between plastids and cytosol that is called the plastid envelope membrane. Many of the known plastid envelope solute transporters integral membrane proteins that catalyze the transport of solutes across biological lipid-bilayer membranes have been identified by biochemical purification and peptide sequencing. This approach is of limited use for less abundant proteins and for proteins of plastid subtypes that are difficult to isolate in preparative amounts. Hence, the majority of plastid envelope membrane transporters are not yet identified at the molecular level. The availability of fully sequenced plant genomes, the progress in bioinformatics research, development, or application of computational tools and approaches to explore biological data such as genome sequences or proteomics data to predict membrane transporters localized in plastids, and the development of highly sensitive proteomics systematic, comprehensive analysis of the full set of proteins (the proteome) in an organism, a specific cell or tissue, or a cellular fraction such as internal membrane systems techniques open new avenues toward identifying additional, to date unknown, plastid envelope membrane transporters.

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.

The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)-specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6-phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6-bisphosphatase. Despite its regulatory role in the feed-forward control of sucrose biosynthesis, variations in the fructose 2,6-bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt-1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1-1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt-1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady-state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.

Cell-wall components from the ectomycorrhizal fungi Amanita muscaria and Hebeloma crustuliniforme and from the spruce pathogen Heterobasidion annosum elicited a transient release of active oxygen species from cultured spruce cells (Picea abies (L.) Karst.). Since the detection of active oxygen was suppressed by catalase, H2O2 was assumed to be the prevailing O2 species. On the other hand, superoxide dismutase enhanced the concentration of detectable H2O2 indicating that the superoxide anion was formed before dismutating to H2O2. The elicitors induced the formation of active oxygen in a dose-dependent manner. Interestingly, elicitors from mycorrhizal fungi had a lower H2O2-inducing activity than equal amounts of cell-wall preparations from the pathogen H. annosum. In Ca(2+)-depleted medium the production of active oxygen by elicitor-treated spruce cells was suppressed. Additionally, the ionophore A 23187 induced active oxygen formation in a medium with Ca(2+) but not in a Ca(2+)-depleted medium. Furthermore, the protein-kinase inhibitor staurosporine inhibited the oxidative burst. At a concentration of 34 nM the effect was diminished to 50%. From these results it is suggested that the release of active oxygen species from cultured spruce cells triggered by cell-wall-derived fungal elicitors depends on external Ca(2+) and a protein-kinase activity. In these respects the effect shows similarities with the well-studied respiratory burst of mammalian neutrophils.