Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo.

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function in vivo Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.

Methane oxidation in anoxic lake water stimulated by nitrate and sulfate addition.

Methanotrophic bacteria play a key role in limiting methane emissions from lakes. It is generally assumed that methanotrophic bacteria are mostly active at the oxic-anoxic transition zone in stratified lakes, where they use oxygen to oxidize methane. Here, we describe a methanotroph of the genera Methylobacter that is performing high-rate (up to 72 µM day-1 ) methane oxidation in the anoxic hypolimnion of the temperate Lacamas Lake (Washington, USA), stimulated by both nitrate and sulfate addition. Oxic and anoxic incubations both showed active methane oxidation by a Methylobacter species, with anoxic rates being threefold higher. In anoxic incubations, Methylobacter cell numbers increased almost two orders of magnitude within 3 days, suggesting that this specific Methylobacter species is a facultative anaerobe with a rapid response capability. Genomic analysis revealed adaptations to oxygen-limitation as well as pathways for mixed-acid fermentation and H2 production. The denitrification pathway was incomplete, lacking the genes narG/napA and nosZ, allowing only for methane oxidation coupled to nitrite-reduction. Our data suggest that Methylobacter can be an important driver of the conversion of methane in oxygen-limited lake systems and potentially use alternative electron acceptors or fermentation to remain active under oxygen-depleted conditions.

Zygotic Genome Activation Occurs Shortly after Fertilization in Maize.

The formation of a zygote via the fusion of an egg and sperm cell and its subsequent asymmetric division herald the start of the plant's life cycle. Zygotic genome activation (ZGA) is thought to occur gradually, with the initial steps of zygote and embryo development being primarily maternally controlled, and subsequent steps being governed by the zygotic genome. Here, using maize (Zea mays) as a model plant system, we determined the timing of zygote development and generated RNA-seq transcriptome profiles of gametes, zygotes, and apical and basal daughter cells. ZGA occurs shortly after fertilization and involves ∼10% of the genome being activated in a highly dynamic pattern. In particular, genes encoding transcriptional regulators of various families are activated shortly after fertilization. Further analyses suggested that chromatin assembly is strongly modified after fertilization, that the egg cell is primed to activate the translational machinery, and that hormones likely play a minor role in the initial steps of early embryo development in maize. Our findings provide important insights into gamete and zygote activity in plants, and our RNA-seq transcriptome profiles represent a comprehensive, unique RNA-seq data set that can be used by the research community.

miR-488-5p and its role in melanoma.

Due to their ability to regulate dozens to hundreds of target genes simultaneously and, therefore, influence several oncogenic pathways at the same time, microRNAs are a fascinating research object in melanoma. MicroRNAs have been identified as regulators of tumor proliferation, invasion and metastasis in melanoma. More precisely, it has been published that dysregulation of miR-488 contibutes to the progression of several cancer entities. However, the biological functions of miR-488, in special miR-488-5p in melanoma, remain unclear. This study showed the involvement of miR-488-5p in Wnt/ß-catenin pathway and the function as a tumor suppressor. Transfection of miR-488-5p mimic led to inhibition of proliferation, migration, anchorage independent growth and led to induction of apoptosis. These data indicated that miR-488-5p acts as a tumor suppressor and is lost during melanoma development. The loss of miR-488-5p was confirmed in vivo by in situ hybridization on melanoma tissue.

FastqPuri: high-performance preprocessing of RNA-seq data.

BACKGROUND: RNA sequencing (RNA-seq) has become the standard means of analyzing gene and transcript expression in high-throughput. While previously sequence alignment was a time demanding step, fast alignment methods and even more so transcript counting methods which avoid mapping and quantify gene and transcript expression by evaluating whether a read is compatible with a transcript, have led to significant speed-ups in data analysis. Now, the most time demanding step in the analysis of RNA-seq data is preprocessing the raw sequence data, such as running quality control and adapter, contamination and quality filtering before transcript or gene quantification. To do so, many researchers chain different tools, but a comprehensive, flexible and fast software that covers all preprocessing steps is currently missing. RESULTS: We here present FastqPuri, a light-weight and highly efficient preprocessing tool for fastq data. FastqPuri provides sequence quality reports on the sample and dataset level with new plots which facilitate decision making for subsequent quality filtering. Moreover, FastqPuri efficiently removes adapter sequences and sequences from biological contamination from the data. It accepts both single- and paired-end data in uncompressed or compressed fastq files. FastqPuri can be run stand-alone and is suitable to be run within pipelines. We benchmarked FastqPuri against existing tools and found that FastqPuri is superior in terms of speed, memory usage, versatility and comprehensiveness. CONCLUSIONS: FastqPuri is a new tool which covers all aspects of short read sequence data preprocessing. It was designed for RNA-seq data to meet the needs for fast preprocessing of fastq data to allow transcript and gene counting, but it is suitable to process any short read sequencing data of which high sequence quality is needed, such as for genome assembly or SNV (single nucleotide variant) detection. FastqPuri is most flexible in filtering undesired biological sequences by offering two approaches to optimize speed and memory usage dependent on the total size of the potential contaminating sequences. FastqPuri is available at https://github.com/jengelmann/FastqPuri . It is implemented in C and R and licensed under GPL v3.

Biosynthesis of Long Chain Alkyl Diols and Long Chain Alkenols in Nannochloropsis spp. (Eustigmatophyceae).

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14-18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes.

Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

Comprehensive Metaboproteomics of Burkitt's and Diffuse Large B-Cell Lymphoma Cell Lines and Primary Tumor Tissues Reveals Distinct Differences in Pyruvate Content and Metabolism.

Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are pathologically and clinically distinct subtypes of aggressive non-Hodgkin B-cell lymphoma. To learn more about their biology, we employed metabolomic and proteomic methods to study both established cell lines as well as cryopreserved and formalin-fixed paraffin-embedded (FFPE) tissue sections of BL and DLBCL. Strikingly, NMR analyses revealed DLBCL cell lines to produce and secrete significantly (padj = 1.72 × 10-22) more pyruvic acid than BL cell lines. This finding could be reproduced by targeted GC/MS analyses of cryopreserved tissue sections of BL and DLBCL cases. Enrichment analysis of an overlapping set of N = 2315 proteins, that had been quantified by nanoLC-SWATH-MS in BL and DLBCL cultured cells and cryosections, supported the observed difference in pyruvic acid content, as glycolysis and pyruvate metabolism were downregulated, while one-carbon metabolism was upregulated in BL compared to DLBCL. Furthermore, 92.1% of the overlapping significant proteins showed the same direction of regulation in cryopreserved and FFPE material. Proteome data are available via ProteomeXchange with identifier PXD004936.

Synergy of interleukin 10 and toll-like receptor 9 signalling in B cell proliferation: Implications for lymphoma pathogenesis.

A network of autocrine and paracrine signals defines B cell homeostasis and is thought to be involved in transformation processes. Investigating interactions of these microenvironmental factors and their relation to proto-oncogenes as c-Myc (MYC) is fundamental to understand the biology of B cell lymphoma. Therefore, B cells with conditional MYC expression were stimulated with CD40L, insulin-like growth factor 1, α-IgM, Interleukin-10 (IL10) and CpG alone or in combination. The impact of forty different interventions on cell proliferation was investigated in MYC deprived cells and calculated by linear regression. Combination of CpG and IL10 led to a strong synergistic activation of cell proliferation (S-phase/doubling of total cell number) comparable to cells with high MYC expression. A synergistic up-regulation of CDK4, CDK6 and CCND3 expression by IL10 and CpG treatment was causal for this proliferative effect as shown by qRT-PCR analysis and inhibition of the CDK4/6 complex by PD0332991. Furthermore, treatment of stimulated MYC deprived cells with MLN120b, ACHP, Pyridone 6 or Ruxolitinib showed that IL10/CpG induced proliferation and CDK4 expression were JAK/STAT3 and IKK/NF-κB dependent. This was further supported by STAT3 and p65/RELA knockdown experiments, showing strongest effects on cell proliferation and CDK4 expression after double knockdown. Additionally, chromatin immunoprecipitation revealed a dual binding of STAT3 and p65 to the proximal promotor of CDK4 after IL10/CpG treatment. Therefore, the observed synergism of IL10R and TLR9 signalling was able to induce proliferation in a comparable way as aberrant MYC and might play a role in B cell homeostasis or transformation.

Gene silencing pathways found in the green alga Volvox carteri reveal insights into evolution and origins of small RNA systems in plants.

BACKGROUND: Volvox carteri (V. carteri) is a multicellular green alga used as model system for the evolution of multicellularity. So far, the contribution of small RNA pathways to these phenomena is not understood. Thus, we have sequenced V. carteri Argonaute 3 (VcAGO3)-associated small RNAs from different developmental stages. RESULTS: Using this functional approach, we define the Volvox microRNA (miRNA) repertoire and show that miRNAs are not conserved in the closely related unicellular alga Chlamydomonas reinhardtii. Furthermore, we find that miRNAs are differentially expressed during different life stages of V. carteri. In addition to miRNAs, transposon-associated small RNAs or phased siRNA loci, which are common in higher land plants, are highly abundant in Volvox as well. Transposons not only give rise to miRNAs and other small RNAs, they are also targets of small RNAs. CONCLUSION: Our analyses reveal a surprisingly complex small RNA network in Volvox as elaborate as in higher land plants. At least the identified VcAGO3-associated miRNAs are not conserved in C. reinhardtii suggesting fast evolution of small RNA systems. Thus, distinct small RNAs may contribute to multicellularity and also division of labor in reproductive and somatic cells.

A statistical approach to virtual cellular experiments: improved causal discovery using accumulation IDA (aIDA).

MOTIVATION: We address the following question: Does inhibition of the expression of a gene X in a cellular assay affect the expression of another gene Y? Rather than inhibiting gene X experimentally, we aim at answering this question computationally using as the only input observational gene expression data. Recently, a new statistical algorithm called Intervention calculus when the Directed acyclic graph is Absent (IDA), has been proposed for this problem. For several biological systems, IDA has been shown to outcompete regression-based methods with respect to the number of true positives versus the number of false positives for the top 5000 predicted effects. Further improvements in the performance of IDA have been realized by stability selection, a resampling method wrapped around IDA that enhances the discovery of true causal effects. Nevertheless, the rate of false positive and false negative predictions is still unsatisfactorily high. RESULTS: We introduce a new resampling approach for causal discovery called accumulation IDA (aIDA). We show that aIDA improves the performance of causal discoveries compared to existing variants of IDA on both simulated and real yeast data. The higher reliability of top causal effect predictions achieved by aIDA promises to increase the rate of success of wet lab intervention experiments for functional studies. AVAILABILITY AND IMPLEMENTATION: R code for aIDA is available in the Supplementary material. CONTACT: franziska.taruttis@ur.de, julia.engelmann@ur.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Causal Modeling of Cancer-Stromal Communication Identifies PAPPA as a Novel Stroma-Secreted Factor Activating NFκB Signaling in Hepatocellular Carcinoma.

Inter-cellular communication with stromal cells is vital for cancer cells. Molecules involved in the communication are potential drug targets. To identify them systematically, we applied a systems level analysis that combined reverse network engineering with causal effect estimation. Using only observational transcriptome profiles we searched for paracrine factors sending messages from activated hepatic stellate cells (HSC) to hepatocellular carcinoma (HCC) cells. We condensed these messages to predict ten proteins that, acting in concert, cause the majority of the gene expression changes observed in HCC cells. Among the 10 paracrine factors were both known and unknown cancer promoting stromal factors, the former including Placental Growth Factor (PGF) and Periostin (POSTN), while Pregnancy-Associated Plasma Protein A (PAPPA) was among the latter. Further support for the predicted effect of PAPPA on HCC cells came from both in vitro studies that showed PAPPA to contribute to the activation of NFκB signaling, and clinical data, which linked higher expression levels of PAPPA to advanced stage HCC. In summary, this study demonstrates the potential of causal modeling in combination with a condensation step borrowed from gene set analysis [Model-based Gene Set Analysis (MGSA)] in the identification of stromal signaling molecules influencing the cancer phenotype.

siPools: highly complex but accurately defined siRNA pools eliminate off-target effects.

Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools-referred to as siPools-containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.

miRA: adaptable novel miRNA identification in plants using small RNA sequencing data.

BACKGROUND: MicroRNAs (miRNAs) are short regulatory RNAs derived from longer precursor RNAs. miRNA biogenesis has been studied in animals and plants, recently elucidating more complex aspects, such as non-conserved, species-specific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can help in computationally identifying genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage from a complex RNA library: while the mature miRNA typically produces many sequence reads, the remaining part of the precursor is covered very sparsely. As recent results suggest, alternative miRNA biogenesis pathways may lead to a more diverse miRNA precursor population than previously assumed. In plants, the latter manifests itself in e.g. complex secondary structures and expression from multiple loci within precursors. Current miRNA identification algorithms often depend on already existing gene annotation, and/or make use of specific miRNA precursor features such as precursor lengths, secondary structures etc. Consequently and in view of the emerging new understanding of a more complex miRNA biogenesis in plants, current tools may fail to characterise organism-specific and heterogeneous miRNA populations. RESULTS: miRA is a new tool to identify miRNA precursors in plants, allowing for heterogeneous and complex precursor populations. miRA requires small RNA sequencing data and a corresponding reference genome, and evaluates precursor secondary structures and precursor processing accuracy; key parameters can be adapted based on the specific organism under investigation. We show that miRA outperforms the currently best plant miRNA prediction tools both in sensitivity and specificity, for data involving Arabidopsis thaliana and the Volvocine algae Chlamydomonas reinhardtii; the latter organism has been shown to exhibit a heterogeneous and complex precursor population with little cross-species miRNA sequence conservation, and therefore constitutes an ideal model organism. Furthermore we identify novel miRNAs in the Chlamydomonas-related organism Volvox carteri. CONCLUSIONS: We propose miRA, a new plant miRNA identification tool that is well adapted to complex precursor populations. miRA is particularly suited for organisms with no existing miRNA annotation, or without a known related organism with well characterized miRNAs. Moreover, miRA has proven its ability to identify species-specific miRNAs. miRA is flexible in its parameter settings, and produces user-friendly output files in various formats (pdf, csv, genome-browser-suitable annotation files, etc.). It is freely available at https://github.com/mhuttner/miRA.

Data Normalization of (1)H NMR Metabolite Fingerprinting Data Sets in the Presence of Unbalanced Metabolite Regulation.

Data normalization is an essential step in NMR-based metabolomics. Conducted properly, it improves data quality and removes unwanted biases. The choice of the appropriate normalization method is critical and depends on the inherent properties of the data set in question. In particular, the presence of unbalanced metabolic regulation, where the different specimens and cohorts under investigation do not contain approximately equal shares of up- and down-regulated features, may strongly influence data normalization. Here, we demonstrate the suitability of the Shapiro-Wilk test to detect such unbalanced regulation. Next, employing a Latin-square design consisting of eight metabolites spiked into a urine specimen at eight different known concentrations, we show that commonly used normalization and scaling methods fail to retrieve true metabolite concentrations in the presence of increasing amounts of glucose added to simulate unbalanced regulation. However, by learning the normalization parameters on a subset of nonregulated features only, Linear Baseline Normalization, Probabilistic Quotient Normalization, and Variance Stabilization Normalization were found to account well for different dilutions of the samples without distorting the true spike-in levels even in the presence of marked unbalanced metabolic regulation. Finally, the methods described were applied successfully to a real world example of unbalanced regulation, namely, a set of plasma specimens collected from patients with and without acute kidney injury after cardiac surgery with cardiopulmonary bypass use.

Increased expression of c-Jun in nonalcoholic fatty liver disease.

Overnutrition is the major cause of nonalcoholic fatty liver disease (NAFLD) and its advanced form nonalcoholic steatohepatitis (NASH). We aimed to develop and characterize a murine model, which resembles both the pathology and nutritional situation, of NASH patients in Western societies. Mice were fed with a NASH-inducing diet (ND) containing sucrose, cholesterol and fats rich in saturated fatty acids in a composition, which mimics Western food. After 12 weeks, ND-fed mice revealed obesity and impaired glucose tolerance. In the liver, ND-feeding led to marked steatosis, hepatocellular damage, inflammation and beginning fibrosis. Transcriptome-wide gene expression analysis and search for over-represented transcription factor target sites among the differentially expressed genes identified activator protein-1 (AP-1) as the most likely factor to cause the transcriptional changes in ND livers. Combining differentially expressed gene and protein-protein interaction network analysis identified c-Jun as hub in the largest connected deregulated sub-network in ND livers. Accordingly, ND livers revealed c-Jun-phosphorylation and nuclear translocation. Moreover, hepatic c-Jun expression was enhanced in ND-fed mice. Combined tissue microarray technology and immunohistochemical analysis confirmed enhanced hepatic c-Jun levels in NAFLD patients, which correlated with inflammation, and notably, with the degree of hepatic steatosis. In summary, our new mouse model shows important pathological changes also found in human NASH and indicates c-Jun/AP-1 activation as critical regulator of hepatic alterations. Abundance of c-Jun in NAFLD likely facilitates development and progression of NASH.

Commonly altered genomic regions in acute myeloid leukemia are enriched for somatic mutations involved in chromatin remodeling and splicing.

Acute myeloid leukemia (AML) is characterized by molecular heterogeneity. As commonly altered genomic regions point to candidate genes involved in leukemogenesis, we used microarray-based comparative genomic hybridization and single nucleotide polymorphism profiling data of 391 AML cases to further narrow down genomic regions of interest. Targeted resequencing of 1000 genes located in the critical regions was performed in a representative cohort of 50 AML samples comprising all major cytogenetic subgroups. We identified 120 missense/nonsense mutations as well as 60 insertions/deletions affecting 73 different genes (∼ 3.6 tumor-specific aberrations/AML). While most of the newly identified alterations were nonrecurrent, we observed an enrichment of mutations affecting genes involved in epigenetic regulation including known candidates like TET2, TET1, DNMT3A, and DNMT1, as well as mutations in the histone methyltransferases NSD1, EZH2, and MLL3. Furthermore, we found mutations in the splicing factor SFPQ and in the nonclassic regulators of mRNA processing CTCF and RAD21. These splicing-related mutations affected 10% of AML patients in a mutually exclusive manner. In conclusion, we could identify a large number of alterations in genes involved in aberrant splicing and epigenetic regulation in genomic regions commonly altered in AML, highlighting their important role in the molecular pathogenesis of AML.

Organic matter degradation in the deep, sulfidic waters of the Black Sea: insights into the ecophysiology of novel anaerobic bacteria.

BACKGROUND: Recent studies have reported the identity and functions of key anaerobes involved in the degradation of organic matter (OM) in deep (> 1000 m) sulfidic marine habitats. However, due to the lack of available isolates, detailed investigation of their physiology has been precluded. In this study, we cultivated and characterized the ecophysiology of a wide range of novel anaerobes potentially involved in OM degradation in deep (2000 m depth) sulfidic waters of the Black Sea. RESULTS: We have successfully cultivated a diverse group of novel anaerobes belonging to various phyla, including Fusobacteriota (strain S5), Bacillota (strains A1T and A2), Spirochaetota (strains M1T, M2, and S2), Bacteroidota (strains B1T, B2, S6, L6, SYP, and M2P), Cloacimonadota (Cloa-SY6), Planctomycetota (Plnct-SY6), Mycoplasmatota (Izemo-BS), Chloroflexota (Chflx-SY6), and Desulfobacterota (strains S3T and S3-i). These microorganisms were able to grow at an elevated hydrostatic pressure of up to 50 MPa. Moreover, this study revealed that different anaerobes were specialized in degrading specific types of OM. Strains affiliated with the phyla Fusobacteriota, Bacillota, Planctomycetota, and Mycoplasmatota were found to be specialized in the degradation of cellulose, cellobiose, chitin, and DNA, respectively, while strains affiliated with Spirochaetota, Bacteroidota, Cloacimonadota, and Chloroflexota preferred to ferment less complex forms of OM. We also identified members of the phylum Desulfobacterota as terminal oxidizers, potentially involved in the consumption of hydrogen produced during fermentation. These results were supported by the identification of genes in the (meta)genomes of the cultivated microbial taxa which encode proteins of specific metabolic pathways. Additionally, we analyzed the composition of membrane lipids of selected taxa, which could be critical for their survival in the harsh environment of the deep sulfidic waters and could potentially be used as biosignatures for these strains in the sulfidic waters of the Black Sea. CONCLUSIONS: This is the first report that demonstrates the cultivation and ecophysiology of such a diverse group of microorganisms from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea. Video Abstract.

Early senescence and cell death in Arabidopsis saul1 mutants involves the PAD4-dependent salicylic acid pathway.

Age-dependent leaf senescence and cell death in Arabidopsis (Arabidopsis thaliana) requires activation of the transcription factor ORESARA1 (ORE1) and is not initiated prior to a leaf age of 28 d. Here, we investigate the conditional execution of events that regulate early senescence and cell death in senescence-associated ubiquitin ligase1 (saul1) mutants, deficient in the PLANT U-BOX-ARMADILLO E3 ubiquitin ligase SAUL1. In saul1 mutants challenged with low light, the switch of age-dependent cell death was turned on prematurely, as indicated by the accumulation of ORE1 transcripts, induction of the senescence marker gene SENESCENCE-ASSOCIATED GENE12, and cell death. However, ORE1 accumulation by itself was not sufficient to cause saul1 phenotypes, as demonstrated by double mutant analysis. Exposure of saul1 mutants to low light for only 24 h did not result in visible symptoms of senescence; however, the senescence-promoting transcription factor genes WRKY53, WRKY6, and NAC-LIKE ACTIVATED BY AP3/PI were up-regulated, indicating that senescence in saul1 seedlings was already initiated. To resolve the time course of gene expression, microarray experiments were performed at narrow intervals. Differential expression of the genes involved in salicylic acid and defense mechanisms were the earliest events detected, suggesting a central role for salicylic acid in saul1 senescence and cell death. The salicylic acid content increased in low-light-treated saul1 mutants, and application of exogenous salicylic acid was indeed sufficient to trigger saul1 senescence in permissive light conditions. Double mutant analyses showed that PHYTOALEXIN DEFICIENT4 (PAD4) but not NONEXPRESSER OF PR GENES1 (NPR1) is essential for saul1 phenotypes. Our results indicate that saul1 senescence depends on the PAD4-dependent salicylic acid pathway but does not require NPR1 signaling.

Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana.

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis-Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.