Components Of Regulatory Networks Research Articles

PACS-1 variant protein is aberrantly localized in C. elegans model of PACS1/PACS2 syndromes.

PACS (Phosphofurin Acidic Cluster Sorting Protein) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of C. elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 co-localize to somatic cytoplasm of many types of cells, and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

Synergistic effects of melatonin and 24-epibrassinolide on chickpea water deficit tolerance

BackgroundWater deficiency stress reduces yield in grain legumes, primarily due to a decrease in the pods number. Melatonin (ML) and 24-epibrassinolide (EBL) are recognized for their hormone-like properties that improve plant tolerance to abiotic stresses. This study aimed to assess the impact of different concentrations of ML (0, 100, and 200 µM) and EBL (0, 3, and 6 µM) on the growth, biochemical, and physiological characteristics of chickpea plants under water-stressed conditions.ResultsThe study's findings indicated that under water-stressed conditions, a decrease in seed (30%) and pod numbers (31%), 100-seed weight (17%), total chlorophyll content (46%), stomatal conductance (33%), as well as an increase in H2O2 (62%), malondialdehyde content (40%), and electrolyte leakage index (40%), resulted in a 40% reduction in chickpea plants grain yield. Our findings confirmed that under water-stressed conditions, seed oil, seed oil yield, and seed protein yield dropped by 20%, 55%, and 36%, respectively. The concurrent exogenous application of ML and EBL significantly reduces oxidative stress, plasma membrane damage, and reactive oxygen species (ROS) content. This treatment also leads to increased yield and its components, higher pigment content, enhanced oil and protein yield, and improved enzymatic and non-enzymatic antioxidant activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase, guaiacol peroxidase, flavonoid, and carotenoid. Furthermore, it promotes the accumulation of osmoprotectants such as proline, total soluble protein, and sugars.ConclusionsOur study found that ML and EBL act synergistically to regulate plant growth, photosynthesis, osmoprotectants accumulation, antioxidant defense systems, and maintain ROS homeostasis, thereby mitigating the adverse effects of water deficit conditions. ML and EBL are key regulatory network components in stressful conditions, with significant potential for future research and practical applications. The regulation metabolic pathways of ML and EBL in water-stressed remains unknown. As a result, future research should aim to elucidate the molecular mechanisms by employing genome editing, RNA sequencing, microarray, transcriptomic, proteomic, and metabolomic analyses to identify the mechanisms involved in plant responses to exogenous ML and EBL under water deficit conditions. Furthermore, the economical applications of synthetic ML and EBL could be an interesting strategy for improving plant tolerance.

Diverse Gene Regulatory Mechanisms Alter Rattlesnake Venom Gene Expression at Fine Evolutionary Scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

Transcription factor expression landscape in Drosophila embryonic cell lines

BackgroundTranscription factor (TF) proteins are a key component of the gene regulatory networks that control cellular fates and function. TFs bind DNA regulatory elements in a sequence-specific manner and modulate target gene expression through combinatorial interactions with each other, cofactors, and chromatin-modifying proteins. Large-scale studies over the last two decades have helped shed light on the complex network of TFs that regulate development in Drosophila melanogaster.ResultsHere, we present a detailed characterization of expression of all known and predicted Drosophila TFs in two well-established embryonic cell lines, Kc167 and S2 cells. Using deep coverage RNA sequencing approaches we investigate the transcriptional profile of all 707 TF coding genes in both cell types. Only 103 TFs have no detectable expression in either cell line and 493 TFs have a read count of 5 or greater in at least one of the cell lines. The 493 TFs belong to 54 different DNA-binding domain families, with significant enrichment of those in the zf-C2H2 family. We identified 123 differentially expressed genes, with 57 expressed at significantly higher levels in Kc167 cells than S2 cells, and 66 expressed at significantly lower levels in Kc167 cells than S2 cells. Network mapping reveals that many of these TFs are crucial components of regulatory networks involved in cell proliferation, cell–cell signaling pathways, and eye development.ConclusionsWe produced a reference TF coding gene expression dataset in the extensively studied Drosophila Kc167 and S2 embryonic cell lines, and gained insight into the TF regulatory networks that control the activity of these cells.

INCREASED EXPRESSION LEVEL OF ADAPTOR PROTEIN Ruk/CIN85 IN DOXORUBICIN-RESISTANT HUMAN NON-SMALL LUNG ADENOCARCINOMA MOR CELLS IS ASSOCIATED WITH THEIR METABOLIC REPROGRAMMING

The aim of the present study was to find out the role of Ruk/CIN85 in modulation of activities/content of key enzymes/components of glycolysis and hydrogen peroxide using as a model human NSCLC MOR wild type and resistant to drugs MOR/0.2R cells. Materials and methods. MOR (ECACC 84112312) and MOR/0.2R (ECACC 96042335), drug-resistant cell line, were cultured in the complete RPMI medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. Using RT2-PCR it was revealed that the level of Ruk/CIN85 mRNA in drug-resistant MOR cells was 10 times higher than in parental MOR cells. The activities of lysyl oxidase (LOX) and diamine oxidase (DAO) were significantly higher in resistant cells It has been shown that these enzymes are associated with aggressiveness of tumor cells. Based on the obtained results, we draw a conclusion that observed changes in the intensity of glycolysis, amine oxidases activities and content of hydrogen peroxide in doxorubicin-resistant MOR/0.2R cells positively correlate with the expression level of the adaptor protein studied. Conclusions. In conclusion, it can be assumed that the adaptor protein Ruk/CIN85 is involved in metabolome reprogramming and may function as an important component of regulatory networks required for the acquisition of drug resistant phenotype by NSCLC cells.

MicroRNA‑190bexpression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4.

MicroRNAs (miRNAs/miRs) are key components of regulatory networks in cancer. Although miR-190b is an important tumor-related miRNA, its role in pancreatic cancer has not been extensively investigated. The aim of the present study was to examine the expression of miR-190b in pancreatic cancer cell lines and tissues and evaluate its effects on cancer progression. Reverse transcription-quantitative PCR (RT-qPCR) analysis was used to measure miR-190b expression levels in human pancreatic cancer cell lines and tissues, and the association between miR-190b expression and clinicopathological characteristics was assessed. An in vitro Transwell invasion assay and an in vivo metastasis formation assay were performed using pancreatic cancer cells. The effect of miR-190b on pancreatic cancer cell proliferation was evaluated using a Cell Counting Kit-8 assay based on an in vivo xenograft mouse model. The direct targets of miR-190b were predicted using bioinformatics tools and were validated through western blotting and luciferase reporter assays. Pancreatic cancer cell lines and tissues were found to express lower levels of miR-190b compared with normal cells and adjacent non-tumor tissues. Furthermore, high expression of miR-190b was found to be positively correlated with low T, N and American Joint Committee on Cancer classifications, and predicted a good prognosis. miR-190b was shown to exert suppressive effects on cancer cell proliferation, invasion and metastasis. In addition, it was also found that miR-190b directly targeted myocyte enhancer factor 2C (MEF2C) and transcription factor 4 (TCF4) in pancreatic cancer, thus serving as a tumor suppressor and a predictor of good prognosis in pancreatic cancer. The immunohistochemistry and RT-qPCR results indicated that the MEF2C and TCF4 expression levels were negatively correlated with the miR-190b expression levels. The findings of the present study highlight the value of miR-190b as a novel target candidate for pancreatic cancer diagnosis and therapy.

Beyond the Genetic Pathways, Flowering Regulation Complexity in Arabidopsis thaliana

Flowering is one of the most critical developmental transitions in plants’ life. The irreversible change from the vegetative to the reproductive stage is strictly controlled to ensure the progeny’s success. In Arabidopsis thaliana, seven flowering genetic pathways have been described under specific growth conditions. However, the evidence condensed here suggest that these pathways are tightly interconnected in a complex multilevel regulatory network. In this review, we pursue an integrative approach emphasizing the molecular interactions among the flowering regulatory network components. We also consider that the same regulatory network prevents or induces flowering phase change in response to internal cues modulated by environmental signals. In this sense, we describe how during the vegetative phase of development it is essential to prevent the expression of flowering promoting genes until they are required. Then, we mention flowering regulation under suboptimal growing temperatures, such as those in autumn and winter. We next expose the requirement of endogenous signals in flowering, and finally, the acceleration of this transition by long-day photoperiod and temperature rise signals allowing A. thaliana to bloom in spring and summer seasons. With this approach, we aim to provide an initial systemic view to help the reader integrate this complex developmental process.

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein–protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile

ABSTRACT Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

Association between long non coding RNA levels and hypertension mediated organ damage

Abstract Purpose Long non coding RNAs (lncRNAs) are emerging as important components of regulatory networks underlying cardiovascular development and pathophysiology. They exhibit distinctive roles in modulating tissue-specific epigenomic states that are critical for the transcriptional and epigenetic reprogramming that may underpin cardiovascular disease pathogenesis. We investigated the association between gene expression levels of the lncRNAs CARMEN and FENDRR in peripheral blood mononuclear cells (PBMCs) and asymptomatic hypertension mediated organ damage. Methods We included 56 patients with essential hypertension (38 women, mean age 77±8 years). Twenty nine normotensive individuals (14 women, mean age 76±10 years) were also included as controls. PBMCs were isolated and lncRNAs' expression levels were determined by quantitative real-time reverse transcription polymerase chain reaction. Hypertension mediated organ damage (MOD), namely carotid wall thickening, left ventricular hypertrophy, and albuminuria were assessed in all patients. Results Hypertensive patients showed significantly higher CARMEN (19.95±5.9 versus 5.69±2.82, p<0.001) and FENDRR (37.7±11.8 versus 10.21±8.02, p<0.001) expression levels compared with controls. Increased CARMEN expression levels in PBMCs showed a significant relationship with the number of organs involved (r=0.49, p=0.001). No significant relationship was revealed for FENDRR levels. Conclusions Our data reveal that in essential hypertension, increased CARMEN expression levels in PBMCs could be a marker of subclinical MOD. Further studies are needed to assess their role as potential biomarkers or future therapeutic targets in those patients. Funding Acknowledgement Type of funding source: None

Identification of common key regulators in rat hepatocyte cell lines under exposure of different pesticides

Pesticides exposure can have harmful effects on human health. The liver is the most common organ of pesticides toxicity due to its major metabolic activity. The molecular mechanism of pesticides effect is complex and is controlled by gene regulatory networks. All components of regulatory networks are controlled by transcription factors and other regulatory elements. Therefore, identification of key regulators through system biology approaches and high-throughput techniques can help to provide comprehensive insights into molecular mechanisms of the pesticide effect. In the current study, a microarray data-set was used to potentially identify molecular mechanisms that regulate gene expression profile of rat hepatocyte cell lines in response to pesticides exposure. Results showed that the number of differentially expressed genes (DEGs) and differentially expressed transcription factors (DE-TFs) were dramatically different among pesticides tested. Results also revealed 205 common DEGs and 11 DE-TFs among pesticides tested. Additionally, we found that six DE-TFs (CREB1, CTNNB1, PPARG, SP1, SRF and STAT3) had the highest number of interactions with other DEGs and acted as the key regulatory genes. The results of this study revealed regulator genes that have the key functions in response to pesticides toxicity in rat liver, which can provide the basis for future studies. Furthermore, these regulatory genes can be used as toxicity biomarkers to improve diagnosis and prognosis.

Challenges of Translating Gene Regulatory Information into Agronomic Improvements.

Improvement of agricultural species has exploited the genetic variation responsible for complex quantitative traits. Much of the functional variation is regulatory, in cis-regulatory elements and trans-acting factors that ultimately contribute to gene expression differences. However, the identification of gene regulatory network components that, when modulated, will increase plant productivity or resilience, is challenging, yet essential to provide increased predictive power for genome engineering approaches that are likely to benefit useful traits. Here, we discuss the opportunities and limitations of using data obtained from gene coexpression, transcription factor binding, and genome-wide association mapping analyses to predict regulatory interactions that impact crop improvement. It is apparent that a combination of information from these data types is necessary for the reliable identification and utilization of important regulatory interactions that underlie complex agronomic traits.

MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants.

Plants, as sessile organisms, adapt to different stressful conditions, such as drought, salinity, extreme temperatures, and nutrient deficiency, via plastic developmental and growth responses. Depending on the intensity and the developmental phase in which it is imposed, a stress condition may lead to a broad range of responses at the morphological, physiological, biochemical, and molecular levels. Transcription factors are key components of regulatory networks that integrate environmental cues and concert responses at the cellular level, including those that imply a stressful condition. Despite the fact that several studies have started to identify various members of the MADS-box gene family as important molecular components involved in different types of stress responses, we still lack an integrated view of their role in these processes. In this review, we analyze the function and regulation of MADS-box gene family members in response to drought, salt, cold, heat, and oxidative stress conditions in different developmental processes of several plants. In addition, we suggest that MADS-box genes are key components of gene regulatory networks involved in plant responses to stress and plant developmental plasticity in response to seasonal changes in environmental conditions.

Integrated transcriptome, small RNA and degradome sequencing approaches provide insights into Ascochyta blight resistance in chickpea.

SummaryAscochyta blight (AB) is one of the major biotic stresses known to limit the chickpea production worldwide. To dissect the complex mechanisms of AB resistance in chickpea, three approaches, namely, transcriptome, small RNA and degradome sequencing were used. The transcriptome sequencing of 20 samples including two resistant genotypes, two susceptible genotypes and one introgression line under control and stress conditions at two time points (3rd and 7th day post inoculation) identified a total of 6767 differentially expressed genes (DEGs). These DEGs were mainly related to pathogenesis‐related proteins, disease resistance genes like NBS‐LRR, cell wall biosynthesis and various secondary metabolite synthesis genes. The small RNA sequencing of the samples resulted in the identification of 651 miRNAs which included 478 known and 173 novel miRNAs. A total of 297 miRNAs were differentially expressed between different genotypes, conditions and time points. Using degradome sequencing and in silico approaches, 2131 targets were predicted for 629 miRNAs. The combined analysis of both small RNA and transcriptome datasets identified 12 miRNA‐mRNA interaction pairs that exhibited contrasting expression in resistant and susceptible genotypes and also, a subset of genes that might be post‐transcriptionally silenced during AB infection. The comprehensive integrated analysis in the study provides better insights into the transcriptome dynamics and regulatory network components associated with AB stress in chickpea and, also offers candidate genes for chickpea improvement.

Evolution of the Symbiosis-Specific GRAS Regulatory Network in Bryophytes.

Arbuscular mycorrhiza is one of the most common plant symbiotic interactions observed today. Due to their nearly ubiquitous occurrence and their beneficial impact on both partners it was suggested that this mutualistic interaction was crucial for plants to colonize the terrestrial habitat approximately 500 Ma ago. On the plant side the association is established via the common symbiotic pathway (CSP). This pathway allows the recognition of the fungal symbiotic partner, subsequent signaling to the nucleus, and initiation of the symbiotic program with respect to specific gene expression and cellular re-organization. The downstream part of the CSP is a regulatory network that coordinates the transcription of genes necessary to establish the symbiosis, comprising multiple GRAS transcription factors (TFs). These regulate their own expression as an intricate transcriptional network. Deduced from non-host genome data the loss of genes encoding CSP components coincides with the loss of the interaction itself. Here, we analyzed bryophyte species with special emphasis on the moss Physcomitrella patens, supposed to be a non-host, for the composition of the GRAS regulatory network components. We show lineage specific losses and expansions of several of these factors in bryophytes, potentially coinciding with the proposed host/non-host status of the lineages. We evaluate losses and expansions and infer clade-specific evolution of GRAS TFs.

LONG NON CODING RNAS IN PERIPHERAL BLOOD MONONUCLEAR CELLS IN HYPERTENSIVES WITH HEART FAILURE WITH PRESERVED EJECTION FRACTION AND THEIR RELATION TO THEIR FUNCTIONAL CAPACITY

Objective: Objective: Long non coding RNAs (lncRNAs) are emerging as important components of regulatory networks underlying cardiovascular development and pathophysiology. They exhibit distinctive roles in modulating tissue-specific epigenomic states that are critical for the transcriptional and epigenetic reprogramming that underpins heart failure (HF) pathogenesis. Our aim was to assess gene expression levels of the lncRNAs CARMEN and FENDRR in peripheral blood mononuclear cells (PBMCs) in hypertensive patients with heart failure with preserved ejection fraction (HFpEF) and to evaluate their association with their exercise capacity. Design and method: Methods: We included 45 patients with essential hypertension and HFpEF (mean age 67 ± 8 years). Twenty one hypertensive patients without HFpEF (mean age 65 ± 12 years) were also included as controls. All patients underwent a cardiopulmonary exercise test (CPXT). PBMCs were isolated and lncRNAs’ expression levels were determined by quantitative real-time reverse transcription polymerase chain reaction Results: Results: Patients with HFpEF showed significantly higher CARMEN (27.93 ± 5.68 versus 11.6 ± 4.8, p = 0.02) and FENDRR (45.72 ± 8.88 versus 16.01 ± 4.04, p = 0.01) expression levels compared with the control group. For hypertensive patients with HFpEF, strong positive correlations were observed between CARMEN expression levels in PBMCs and peakVO2 and (r = 0.46, p = 0.001), VE/VCO2 (r = 0.45, p = 0.002) as well as exercise duration (r = 0.427, p = 0.003). We also observed a weak but significant negative correlation between FENDRR expression levels in PBMCs and VE/VCO2 (r = -0.35, p = 0.02). Conclusions: Conclusions: Our data reveal that CARMEN and FENDRR expression levels in PBMCs of hypertensive patients with HFpEF may be related to their functional capacity. Further studies are needed to assess their role as therapeutic targets in those patients

Noncoding RNAs in the Regulation of Pluripotency and Reprogramming.

Pluripotent stem cells have great potential for developmental biology and regenerative medicine. Embryonic stem cells, which are obtained from blastocysts, and induced pluripotent stem cells, which are generated by the reprogramming of somatic cells, are two main types of pluripotent cells. It is important to understand the regulatory network that controls the pluripotency state and reprogramming process. Various types of noncoding RNAs (ncRNAs) have emerged as substantial components of regulatory networks. The most studied class of ncRNAs in the context of pluripotency and reprogramming is microRNAs (miRNAs). In addition to canonical microRNAs, other types of small RNAs with miRNA-like function are expressed in PSCs. Another class of ncRNAs, long ncRNAs, are also involved in pluripotency and reprogramming regulation. Thousands of ncRNAs have been annotated to date, and a significant number of the molecules do not have known function. In this review, we briefly summarized recent advances in this field and described existing genome-editing approaches to study ncRNA functions.

Identification and characterization of regulatory network components for anthocyanin synthesis in barley aleurone

BackgroundAmong natural populations, there are different colours of barley (Hordeum vulgare L.). The colour of barley grains is directly related to the accumulation of different pigments in the aleurone layer, pericarp and lemma. Blue grain colour is due to the accumulation of anthocyanins in the aleurone layer, which is dependent on the presence of five Blx genes that are not sequenced yet (Blx1, Blx3 and Blx4 genes clustering on chromosome 4HL and Blx2 and Blx5 on 7HL). Due to the health benefits of anthocyanins, blue-grained barley can be considered as a source of dietary food. The goal of the current study was to identify and characterize components of the anthocyanin synthesis regulatory network for the aleurone layer in barley.ResultsThe candidate genes for components of the regulatory complex MBW (consisting of transcription factors MYB, bHLH/MYC and WD40) for anthocyanin synthesis in barley aleurone were identified. These genes were designated HvMyc2 (4HL), HvMpc2 (4HL), and HvWD40 (6HL). HvMyc2 was expressed in aleurone cells only. A loss-of-function (frame shift) mutation in HvMyc2 of non-coloured compared to blue-grained barley was revealed. Unlike aleurone-specific HvMyc2, the HvMpc2 gene was expressed in different tissues; however, its activity was not detected in non-coloured aleurone in contrast to a coloured aleurone, and allele-specific mutations in its promoter region were found. The single-copy gene HvWD40, which encodes the required component of the regulatory MBW complex, was expressed constantly in coloured and non-coloured tissues and had no allelic differences. HvMyc2 and HvMpc2 were genetically mapped using allele-specific developed CAPS markers developed. HvMyc2 was mapped in position between SSR loci XGBS0875-4H (3.4 cM distal) and XGBM1048-4H (3.4 cM proximal) matching the region chromosome 4HL where the Blx-cluster was found. In this position, one of the anthocyanin biosynthesis structural genes (HvF3’5’H) was also mapped using an allele-specific CAPS-marker developed in the current study.ConclusionsThe genes involved in anthocyanin synthesis in the barley aleurone layer were identified and characterized, including components of the regulatory complex MBW, from which the MYC-encoding gene (HvMyc2) appeared to be the main factor underlying variation of barley by aleurone colour.

Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae.

BackgroundThe gene expression and secretion of fungal lignocellulolytic enzymes are tightly controlled at the transcription level using independent mechanisms to respond to distinct inducers from plant biomass. An advanced systems-level understanding of transcriptional regulatory networks is required to rationally engineer filamentous fungi for more efficient bioconversion of different types of biomass.ResultsIn this study we focused on ten chemically defined inducers to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae and shed light on the complex network of transcriptional activators required. The chemical diversity analysis of the inducers, based on 186 chemical descriptors calculated from the structure, resulted into three clusters, however, the global, metabolic and extracellular protein transcription of the A. oryzae genome were only partially explained by the chemical similarity of the enzyme inducers. Genes encoding enzymes that have attracted considerable interest such as cellobiose dehydrogenases and copper-dependent polysaccharide mono-oxygenases presented a substrate-specific induction. Several homology-model structures were derived using ab-initio multiple threading alignment in our effort to elucidate the interplay of transcription factors involved in regulating plant-deconstructing enzymes and metabolites. Systematic investigation of metabolite-protein interactions, using the 814 unique reactants involved in 2360 reactions in the genome scale metabolic network of A. oryzae, was performed through a two-step molecular docking against the binding pockets of the transcription factors AoXlnR and AoAmyR. A total of six metabolites viz., sulfite (H2SO3), sulfate (SLF), uroporphyrinogen III (UPGIII), ethanolamine phosphate (PETHM), D-glyceraldehyde 3-phosphate (T3P1) and taurine (TAUR) were found as strong binders, whereas the genes involved in the metabolic reactions that these metabolites appear were found to be significantly differentially expressed when comparing the inducers with glucose.ConclusionsBased on our observations, we believe that specific binding of sulfite to the regulator of the cellulase gene expression, AoXlnR, may be the molecular basis for the connection of sulfur metabolism and cellulase gene expression in filamentous fungi. Further characterization and manipulation of the regulatory network components identified in this study, will enable rational engineering of industrial strains for improved production of the sophisticated set of enzymes necessary to break-down chemically divergent plant biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0224-5) contains supplementary material, which is available to authorized users.

Conserved Noncoding Sequences Highlight Shared Components of Regulatory Networks in Dicotyledonous Plants

Conserved noncoding sequences (CNSs) in DNA are reliable pointers to regulatory elements controlling gene expression. Using a comparative genomics approach with four dicotyledonous plant species (Arabidopsis thaliana, papaya [Carica papaya], poplar [Populus trichocarpa], and grape [Vitis vinifera]), we detected hundreds of CNSs upstream of Arabidopsis genes. Distinct positioning, length, and enrichment for transcription factor binding sites suggest these CNSs play a functional role in transcriptional regulation. The enrichment of transcription factors within the set of genes associated with CNS is consistent with the hypothesis that together they form part of a conserved transcriptional network whose function is to regulate other transcription factors and control development. We identified a set of promoters where regulatory mechanisms are likely to be shared between the model organism Arabidopsis and other dicots, providing areas of focus for further research.