Browsing by Subject "Gene expression"

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Ammonium uptake, mediated by ammonium transporters, mitigates manganese toxicity in duckweed, Spirodela polyrhiza
(2023) Kishchenko, Olena; Stepanenko, Anton; Straub, Tatsiana; Zhou, Yuzhen; Neuhäuser, Benjamin; Borisjuk, Nikolai
Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism.
Analyse komplexer Merkmale beim Schwein mittels SNP-Chip Genotypen, Darmmikrobiota- und Genexpressionsdaten
(2017) Maushammer, Maria; Bennewitz, Jörn
In the present scientific research, SNP chip genotypes, gut microbiota and gene expression data were used for analysing complex traits in a Piétrain population. These data were collected from around 200 performance tested sows and were used for genetic and microbial analyses of complex trait as well as for structural and functional meat quality traits. The gut microbiome plays a major role in the immune system development, state of health and energy supply of the host. Quantitative-genetic methods were applied to analyse the interrelationship between pig gut microbiota compositions, complex traits (daily gain, feed conversion and feed intake) and pig genomes. The specific aims were to characterize the gut microbiota of the pigs, to analyse the effects of host genetics on gut microbial composition, and to investigate the role of gut microbial composition on the host’s complex traits. The pigs were genotyped with a standard 60K SNP chip. Microbial composition was characterized by 16S rRNA gene amplicon sequencing technology. Ten out of 51 investigated bacterial genera showed a significant host heritability, ranging from 0.32 to 0.57. Conducting genome wide association analysis showed associations of 22 SNPs and six bacterial genera. The potential candidate genes identified are involved in the immune system, mucosa structure and secretion of digestive juice. These results show, that parts of the gut microbiota are heritable and that the gut microbiome can be seen as quantitative trait. Microbial mixed linear models were applied to estimate the microbiota variance for each of the investigated traits. The fraction of phenotypic variance explained by the microbial variance was 0.28, 0.21, and 0.16 for daily gain, feed conversion, and feed intake, respectively. The SNP data and the microbiota data were used to predict the phenotypes of the traits using both, genomic best linear unbiased prediction (G-BLUP) and microbial best linear unbiased prediction (M-BLUP) methods. The prediction accuracies of G-BLUP were 0.35, 0.23, and 0.20 for daily gain, feed conversion, and feed intake, respectively. The corresponding prediction accuracies of M-BLUP were 0.41, 0.33, and 0.33. Thus, the gut microbiota can be seen as an explaining variable for complex traits like daily gain, feed conversion and feed intake. In addition, in combination with meat quality traits, transcript levels of muscle tissue were analysed at time of slaughtering. This study should give an insight into the biological processes involved in meat quality characteristics. The aims were to functionally characterise differentially expressed genes, to link the functional information with structural information obtained from GWAS, and to identify potential candidate genes based on these results. An important meat quality trait is the intramuscular fat content, since it affects the juiciness, the taste and the tenderness of the meat. Another important trait is drip loss which causes not only a loss of weight but also a loss of important proteins. Both traits have an impact on the consumer acceptance of fresh meat products. For each of the two traits, eight discordant sibling pairs were selected out of the Piétrain sample and were used for genome-wide gene expression analyses. Thirty five and 114 genes were identified as differentially expressed and trait correlated genes for intramuscular fat content and drip loss, respectively. On the basis of functional annotation, gene groups belonging to the energy metabolism of the mitochondria, the immune response and the metabolism of fat, were associated with intramuscular fat content. Gene groups associated with protein ubiquitination, mitochondrial metabolism, and muscle structural proteins were associated with drip loss. Furthermore, genome-wide association analyses were carried out for these traits and their results were linked to the genome-wide expression analysis by functional annotation. In this context, intramuscular fat was related to muscle contraction, transmembrane transport and nucleotide binding. Drip loss was characterized by the endomembrane system, the energy generation of cells, and phosphorus metabolic processes. Three and four potential candidate genes were identified for intramuscular fat content and drip loss, respectively.
Broad time‐dependent transcriptional activity of metabolic genes of E. coli O104:H4 strain C227/11Φcu in a soil microenvironment at low temperature
(2023) Detert, Katharina; Währer, Jonathan; Nieselt, Kay; Schmidt, Herbert
In the current study, metabolic genes and networks that influence the persistence of pathogenic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soil microenvironments at low temperature were investigated. The strain was incubated in alluvial loam (AL) and total RNA was prepared from samples at time point 0, and after 1 and 4 weeks. Differential transcriptomic analysis was performed by RNA sequencing analysis and values obtained at weeks 1 and 4 were compared to those of time point 0. We found differential expression of more than 1500 genes for either time point comparison. The two lists of differentially expressed genes were then subjected to gene set enrichment of Gene Ontology terms. In total, 17 GO gene sets and 3 Pfam domains were found to be enriched after 1 week. After 4 weeks, 17 GO gene sets and 7 Pfam domains were statistically enriched. Especially stress response genes and genes of the primary metabolism were particularly affected at both time points. Genes and gene sets for uptake of carbohydrates, amino acids were strongly upregulated, indicating adjustment to a low nutrient environment. The results of this transcriptome analysis show that persistence of C227/11Φcu in soils is associated with a complex interplay of metabolic networks.
The functional diversity of the high-affinity nitrate transporter gene family in hexaploid wheat: Insights from distinct expression profiles
(2023) Sigalas, Petros P.; Buchner, Peter; Kröper, Alex; Hawkesford, Malcolm J.
High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies.
Metabolomic and gene expression studies reveal the diversity, distribution and spatial regulation of the specialized metabolism of yacón (Smallanthus sonchifolius, Asteraceae)
(2020) Padilla-González, Guillermo F.; Amrehn, Evelyn; Frey, Maximilian; Gómez-Zeledón, Javier; Kaa, Alevtina; Da Costa, Fernando B.; Spring, Otmar
Smallanthus sonchifolius, also known as yacón, is an Andean crop species commercialized for its nutraceutical and medicinal properties. The tuberous roots of yacón accumulate a diverse array of probiotic and bioactive metabolites including fructooligosaccharides and caffeic acid esters. However, the metabolic diversity of yacón remains unexplored, including the site of biosynthesis and accumulation of key metabolite classes. We report herein a multidisciplinary approach involving metabolomics, gene expression and scanning electron microscopy, to provide a comprehensive analysis of the diversity, distribution and spatial regulation of the specialized metabolism in yacón. Our results demonstrate that different metabolic fingerprints and gene expression patterns characterize specific tissues, organs and cultivars of yacón. Manual inspection of mass spectrometry data and molecular networking allowed the tentative identification of 71 metabolites, including undescribed structural analogues of known bioactive compounds. Imaging by scanning electron microscopy revealed the presence of a new type of glandular trichome in yacón bracts, with a distinctive metabolite profile. Furthermore, the high concentration of sesquiterpene lactones in capitate glandular trichomes and the restricted presence of certain flavonoids and caffeic acid esters in underground organs and internal tissues suggests that these metabolites could be involved in protective and ecological functions. This study demonstrates that individual organs and tissues make specific contributions to the highly diverse and specialized metabolome of yacón, which is proving to be a reservoir of previously undescribed molecules of potential significance in human health.
Nutritional regulation of DNA methylation and gene expression in maize
(2018) Mager, Svenja; Ludewig, Uwe
DNA methylation in plants plays a role in transposon silencing, genome stability and gene expression regulation. Environmental factors alter the methylation pattern of DNA and recently nutrient stresses, such as phosphate starvation, were shown to alter DNA methylation. DNA methylation had been frequently addressed in plants with notably small genomes that are poor in transposons. Here, part of the DNA methylome of nitrogen-, phosphorus- and zinc-deficient (-N, -P and -Zn, respectively) maize roots were compared by reduced representation sequencing and their relationship with gene expression under prolonged stresses analyzed. Tremendous DNA methylation loss was encountered in maize under nitrogen and zinc deficiency, but much less under phosphorus deficiency. This occurred only in the symmetrical cytosine contexts, predominantly in CG context, but also in the CHG context. In contrast to other plants, differential methylation in the more flexible CHH context was essentially absent. For each sample, specific nutrient deficiency-regulated genes were differentially expressed. In -Zn samples the lowest number of differentially expressed genes was found while -N and -P samples contained a similar number of differentially expressed genes. For all samples, differentially methylated regions (DMRs) were predominantly identified in transposable elements (TEs). A minor fraction of such DMRs was associated with altered gene expression of nearby genes in -N and -P. Interestingly, although these TEs were mostly hypomethylated, they were associated with both upand down-regulated gene expression. For -Zn, these associations were not found but a correlation between hypomethylation of gene bodies and expression of some genes. Here again, hypomethylation occurred with up- and downregulation of gene expression. The results suggested a different methylome regulation in maize compared to rice and Arabidopsis upon nutrient deficiencies indicating a nutrient- and species-specific association of genomic DNA methylation and gene expression. The limited correlation between differential DNA methylation and gene expression suggested that heritable regulation of the expression of nutrient deficiency-regulated genes was not the primary function of the methylation loss. Rather, the major function of the DNA methylation loss in this experiment may have been to increase the genetic diversity in the next generation by increased frequency of recombination events, mutations and transposable element movements.
Regulatory elements controlling the expression of OR37 genes
(2007) Zhang, Yongquan; Breer, Heinz
The genes of the OR37 family are clustered in two loci (cluster I and cluster II) on mouse chromosome 4. These genes encode distinct olfactory receptors (ORs) which are characterised by an insertion of six amino acids in the third extracellular loop and moreover, these receptor types are only expressed in cells which are segregated in a small patch on the central nasal turbinate. As first steps to unravel the molecular basis of this unique topographic expression pattern previous studies have led to the identification of highly conserved sequence motifs including an olf-1 site in the putative promoter region of these genes and subsequently several transcription factors were identified which did bind to these sites. However, it remained elusive if an interaction between the transcription factors and the putative promoter sites may have functional implications. Therefore, a heterologous system was employed to assess the consequence of an interaction between the putative promoters and the transcription factors. HEK 293 cells were cotransfected with a reporter gene under the control of putative mOR37 promoter regions and an expression vector based gene encoding the transcription factor. The expression rate of the reporter gene was monitored by measuring luciferase activity. It was found that the three O/E transcription factors (O/E-1, O/E-2 and O/E-4) induced significant activation of the mOR37 promoters; in addition, it was observed that the putative promoters of other OR genes were also activated, suggesting that the O/E proteins may play a general role in the regulation of OR gene expression. Mutagenesis experiments revealed that the effects of O/E proteins were dependent on the presence of an olf-1 site within the promoter region. For the transcription factor Lhx-2 it was found that not all but only promoters of distinct OR-genes were affected. For the mOR37 promoters a simultaneous action of O/E protein and Lhx-2 elicited an increase of reporter gene expression. The data indicate that the putative mOR37 promoters could drive gene expression in the presence of the crucial transcription factors in this heterologous system. In order to explore to what extent the promoter may contribute to the characteristic topographic expression pattern of the mOR37 genes in vivo, a mOR37C transgene which included the coding exon and the putative promoter, was randomly inserted into the mouse genome. Seven lines were obtained; in all lines the transgene was specifically expressed in olfactory sensory neurons (OSN). In six lines the transgene expression was restricted to the central patch of the olfactory turbinates, typical for the OR37 genes. In one line (line 7) the transgene was also expressed in OSNs ectopically positioned outside the patch within the medial zone. It was found that the transgene was expressed in a mutually exclusive manner and from only one allele. The axons of OSNs expressing the transgene co-converged in the same glomerulus with the axons from neurons expressing the endogenous gene. In line #7 the formation of ectopic glomeruli was observed. The number of OSNs expressing the transgene varied considerably among lines; these differences were independent from the copy number of the transgene. The data indicate that the short putative promoters, most likely the conserved motifs, were sufficient to drive the OR37 gene expression in a tissue specific way and most aspects of the OR37 gene expression were mimicked by the transgene; however, considerable differences between certain lines suggested additional regulatory elements, such as a locus control region (LCR). Since regulatory elements for gene transcription, such as promoters, enhancers and LCRs, appear to be conserved across species, a comparative approach was utilized to search for the LCR-like element for the OR37 locus by sequence alignment across distantly related mammals. A segment of 270 base pairs located 137 Kb upstream of OR37 cluster I was found to be highly conserved between mouse, human, dog and opossum. It was not associated with an exon of any known gene and was highly correlated with OR37 cluster I rather than with the neighboring genes, since the flanking genes did not show syntenic conservation in the opossum genome. A homologous counterpart for this segment was found downstream of the OR37 cluster II locus; an alignment of the cluster II sequence across species identified the conservation of this counterpart. Examination for relevant motifs in this segment and comparison with the conserved H element revealed two common transcription factor binding sites, at least one of them is known to be essential for generating DNase I hypersensitive sites in the LCR of the beta globin gene locus. Further studies are required to evaluate a possible role of this conserved segment in the regulation of the OR37 gene expression.
Successful silencing of the mycotoxin synthesis gene TRI5 in fusarium culmorum and observation of reduced virulence in VIGS and SIGS experiments
(2022) Tretiakova, Polina; Voegele, Ralf Thomas; Soloviev, Alexander; Link, Tobias Immanuel
Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53–85% and 61–87%, respectively, and reduced TRI5 expression by 84–97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.
The genetic basis of heat tolerance in temperate maize (Zea mays L.)
(2016) Frey, Felix P.; Stich, Benjamin
The global mean temperature and probability of heat waves are expected to increase in the future, which has the potential to cause severe damages to maize production. To elucidate the genetic mechanisms of the response of temperate maize to heat stress and for the tolerance to heat stress, in a first experiment I applied gene expression profiling. Therewith, I investigated the transcriptomic response of temperate maize to linearly increasing heat levels. Further, I identified genes associated with heat tolerance in a set of eight genotypes with contrasting heat tolerance behavior. I identified 607 heat responsive genes, which elucidate the genetic pathways behind the response of maize to heat stress and can help to expand the knowledge of plant responses to other abiotic stresses. Further, I identified 39 genes which were differentially regulated between heat tolerant and heat susceptible inbreds and, thus, are putative heat tolerance candidate genes. Two of these candidate genes were located in genome regions which were associated with heat tolerance during seedling and adult stage that have been detected in QTL studies in the frame of this thesis. Their exact molecular functions, however, are still unknown. The statistical approach to identify heat tolerance genes, presented in my thesis, enables researchers to investigate the transcriptomic response of multiple genotypes to changing conditions across several experiments, considering their natural variation for a quantitative trait. In order to develop more heat tolerant cultivars, knowledge of natural variation for heat tolerance in temperate maize is indispensable. Therefore, heat tolerance was assessed in a set of intra- and interpool Dent and Flint populations on a multi-environment level. Usually, heat stress in temperate Europe occurs during the adult stage of maize. However, as maize is of increasing importance as a biogas crop, farmers can reduce the growth period by postponed sowing after the harvest of the winter cereals in early summer and, thus, sensitive maize seedlings can be exposed to heat stress. Therefore, I aimed to assess heat tolerance in six connected segregating Dent and Flint populations during both developmental stages considering besides multiple environments also multiple traits. At heat stress, I observed an average decrease of 20% of the shoot dry weight during seedling stage and an average of 50% of yield loss, when heat stress was present during adult stage. At the heat locations heat stress was present in the year, when the experiments were conducted as temperatures exceeded 32°C there for more than 400 hours during the growing period in contrast to less than 30 hours at the standard locations. This emphasizes that maize crop production can suffer with the increasing number and intensity of summer heat waves. Furthermore, the study revealed strong differences between genotypes, which was indispensable to differentiate between heat tolerant and heat susceptible inbred lines. The tested genotypes originating from the Flint pool turned out to possess higher heat tolerance during seedling stage, whereas the genotypes derived from the Dent pool possessed higher heat tolerance during adult stage. This fact could be exploited by the maintenance of two pools with contrasting heat tolerance and could be beneficial for hybrid breeding. A direct selection of more heat tolerant genotypes in terms of grain yield is expensive and time-consuming. To facilitate the selection process in order to develop more heat tolerant cultivars, breeders could make use of marker assisted selection. To lay the foundation for this technique, in my thesis, QTL for heat tolerance during adult and during seedling stage were identified with the previously mentioned populations. Two QTL explained 19% of the total variance for heat tolerance with respect to grain yield in a simultaneous fit. Furthermore each two QTL were identified for two principal components, which accounted for heat tolerance during seedling stage. They explained 14 and 12% of the respective variance. The results can be used by breeding companies to develop marker assays in order to select heat tolerant genotypes from their proprietary genetic material during both stages in an initial screening. This would reduce the field capacities considerably, which are needed to test heat tolerance on a field level.
The wheat AMT2 (AMmonium Transporter) family, possible functions in ammonium uptake and pathogenic/symbiotic interactions
(2023) Porras‐Murillo, Romano; Zhao, Yufen; Hu, Jinling; Ijato, Toyosi; Retamal, Joseline Palafox; Ludewig, Uwe; Neuhäuser, Benjamin
Ammonium uptake into wheat roots relies primarily on two AMmonium Transporters of subfamily one, while the wheat genome comprises 4 to 6 AMT2 type transporters. Plant AMT2s generally show functions in root‐to‐shoot translocation or pathogenic and symbiotic plant–microorganism interactions. We addressed the activity of TaAMT2s in ammonium transport. Nitrogen‐dependent expression implicated a physiological function in ammonium uptake for TaAMT2;1 and in ammonium distribution for TaAMT2;2‐6.
Wirt-Virus Wechselwirkungen bei der Infektion durch Acanthocystis turfacea Chlorella Virus 1 : Regulation der Genexpression früher Gene und des Ubiquitin-Systems
(2021) Lindner, Kamila; Pfitzner, Artur J. P.
Acanthocystis turfacea Chlorella Virus 1 (ATCV-1) is a virus of the genus Chloroviruses that infects the unicellular green alga Chlorella heliozoae. The infection with ATCV-1 is lethal for the algae and requires the correct expression of the 860 hypothetical virus genes. These genes are divided into early, early/late and late genes which are expressed at different times, depending on their protein function. Gene expression is regulated by their corresponding viral promoters and can be controlled by viral or host-specific transcription factors. The stability of proteins is regulated by the host’s ubiquitin system. This study investigated the expression of early viral genes and the ubiquitin- mediated protein degradation regulated by ATCV-1. By investigating the promoters of the early genes of ATCV-1 Z174L, Z765R and Z798L respectively, consensus sequences were identified including a Hex motif and a TATA box that can be bound by viral, but most importanly, host transcription factors. By in vivo interactions with G-box binding factors, a direct regulation of the early promoters, including aforementioned Hex motif, could be demonstrated. Along with this new regulatory mechanism for the expression of early viral genes, evidence for additional mechanisms for the regulation of early genes with different consensus sequences, such as AATGACA, were found. In the second part of this study, three novel viral proteins were identified as proteins of the ubiquitin system: a viral ubiquitin (Z203L), a viral RING E3 ligase (Z292L) and a viral SKP1 protein (Z339L). Experiments have shown that ATCV-1 is able to interfere with the host’s ubiquitin system using these proteins. Although the E3 ligases are responsible for the specific ubiquitination of the target proteins (e.g. Z292L in the case of enolase), the additional expression of a viral ubiquitin ensures a sufficient amount of the signal protein is present. The results of this study demonstrate a wide range of host-virus interactions at the level of gene regulation and protein degradation. ATCV-1 can use host factors to initiate its own gene expression and, with the help of the components of the ubiquitin system encoded in the viral genome, reprogram the host’s protein degradation.