Browsing by Subject "Transkriptom"

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Adaptations of maize to low phosphate availability : establishing regulatory networks from large-scale quantitative proteomic profiling
(2022) He, Mingjie; Schulze, Waltraud
Maize (Zea mays) is an important crop in global for human food, animal feed and industrial usage. Suboptimal phosphorus (P) availability is one of the primary constraints for maize growth and productivity (Jianbo Shen et al., 2011; L.pez-Arredondo et al., 2014). Over 70% arable land suffers from P-deficiency, and plants can take up small amounts of P from the soil due to P-fixation. However, over-application of P fertilizer has frequently happened in last decades and resulted in environmental pollution (L.pez- Arredondo et al., 2014). Modern agriculture calls for maintaining productivity while reducing synthetic-P fertilizer inputs and losses, thus, requiring breeding of novel cultivars to increase phosphate use efficiency (PUE) (Balemi and Negisho, 2012; X., Li, Mang, et al., 2021; Mardamootoo et al., 2021). Understanding the regulation of maize to low phosphate(LP)-availability at the molecular level will offer unlimited potential for the development of selection markers and engineering targets in breeding programs. Nowadays, “OMIC” approaches and computational science are developing rapidly. They are advanced tools for investigation of molecular adaptations on a large-scale and in a systemic view. Thereby, the major research task within this thesis is to reveal P-deficiency induced responsive components and regulations at protein level based on proteomic profiles, aiming to provide promising candidate genes/proteins for research on the molecular mechanisms of adaptation to LP-stress, and potentially to provide promising candidate gene/proteins for development of selection markers and engineering targets to obtain desired traits, in the long term goal of improving PUE in novel cultivars. In Chapter 1, we focused on six genotypes (EP1, F2, F142, F160, SF1, SM1) with close genetic background but several contrasting traits to LP-stress, such as PUE (X., Li, Mang, et al., 2021). They were cultured in pot with either sufficient or inefficient P-fertilizer in a climate chamber for one month. The young seedlings were sampled by root and shoot for analysis of multiple traits, transcriptome and proteome. Firstly, we constructed the co-expression network of proteins and transcripts separately using WGCNA method (Langfelder and Horvath, 2008), which predicted potential protein-protein interactions or their co-regulations. Secondly, we categorized proteins/transcripts to modules according to their different coexpression patterns, thus, identified potential determining relationships of modules-traits. Thirdly, we compared the responses between transcripts and proteins, presenting their responses being concordant or dis-concordant. Fourthly, we identified common and genotype-specific P-starvation response modules and biological processes. Finally, we focused on protein kinases, which play roles as regulators, to demonstrated protein kinases-centered network and validated protein interactions between mitogenactivated protein kinase-kinase 1 (MEK1, Zm00001d043609) either with sucrose synthase1 (SH1,Zm00001d045042) or translation elongation factor 1-gamma 3 (eEF1B-γ, Zm00001d046352). MEK1 is a potential genotype-specific regulator via sucrose metabolism and translation elongation process. In Chapter 2, we aimed to adapted an experimental workflow for phosphoproteome analysis in maize, addressing the interference to phosphoproteome quantification by fibers, secondary metabolites and low abundant of phosphorylated proteins. In this manuscript, we described a rapid and universal protocol for both proteome and phosphoproteome analysis that is suitable for cereal crops. The results of phosphoproteome in maize root testing samples showed that proteins within kinase-centered network in Chapter 1 can be largely quantified based on this workflow. It provides a possible way to analyze phosphorylation dynamics to P-starvation responses, it allows further investigation for kinase-centered 1 network in Chapter 1 to identify phosphorylation pairs of “protein kinase – protein substrate”, which will largely expand a view on P-starvation regulations through posttranslational modifications.
Identification and analysis of a transcriptome of Douglas-fir (Pseudotsuga menziesii) and population structure inference using different next-generation sequencing techniques
(2015) Müller, Thomas; Schmid, Karl J.
Predictions assume severe changes in the climatic conditions in Central Europe in the coming decades. Longer periods of drought and less precipitation during summer are expected. Plants cannot change their habitat and have to adapt to the new conditions or their offspring has to colonize new ecological niches. Due to the long generation times in trees it is important to know if and how trees can cope with the expected climatic conditions. Forest managers already give thought to the composition of future forests, because they have to choose species and populations which have no or only few problems with the changed climate. Douglas-fir (Pseudotsuga menziesii) is a promising tree species for this purpose, because it is adapted to different habitats and climate zones in its natural distribution range in North America. The two main varieties, coastal and interior Douglas-fir, differ genotypically and phenotypically, e.g. in drought tolerance. Douglas-fir trees, mainly of the coastal variety, showed good growth performances in field trials in Germany. Hence, a research project called "DougAdapt" was designed to analyze and to link genotypic and phenotypic differences in several coastal and interior Douglas-fir provenances. In this project, trees from field trials and from greenhouse experiments were sampled. To analyze the genetic diversity of the provenances we first generated reference sequences. Even with modern and cost-efficient next-generation sequencing technologies it would be very expensive to decipher the ~ 19 gigabases of the Douglas-fir genome completely. An alternative to whole genome sequencing is transcriptome sequencing, in which only the coding regions of a genome are sequenced. The transcriptome sequencing, which was performed for the first time in Douglas-fir, resulted in a large number of putative unique transcripts (PUTs). Comparisons with published transcriptomes of other plant species showed that the PUTs represented the transcriptome of Douglas-fir comprehensively. As the sampled seedlings were part of a drought stress experiment and grew under controlled conditions, we were able to identify drought related candidate PUTs, which may be part of the trees response to drought. Furthermore, more than 27,000 previously unknown single nucleotide polymorphisms (SNPs) in Douglas-fir could be identified. SNPs can influence the phenotype of individuals, and they can be used for instance as markers or to analyze genetic diversity. The analysis of genetic diversity of Douglas-fir provenances and the search for genes which may be part of local adaptation were performed with a sequence capture experiment. In sequence capture only predefined regions of a genome are sequenced. We showed that sequence capture based on PUTs as target regions is applicable in species with large and mostly unknown genomes. The polymorphic drought related candidate PUTs showed higher genetic differentiation than the remaining genes. Nevertheless, none of them was among the candidate PUTs for positive selection, which in turn are probably part of the local adaptation of the trees. Despite a high level of gene flow between coastal and interior provenances, the SNP data showed genetic differentiation between both varieties but only very low differentiation between the coastal provenances. We also investigated if genotyping-by-sequencing (GBS) is a suitable method to detect polymorphisms in Douglas-fir and compared the results of two GBS experiments with the sequence capture. The genome is digested with one or several restriction enzymes in GBS. Afterwards, only fragments with a specific length are sequenced, which considerably reduces the part of the genome that is sequenced as well as the costs. The advantage compared to sequence capture is the possibility to sample more individuals at the same time with less effort and costs. We showed that a digestion with two restriction enzymes results in more SNPs with less missing data, compared to using only one restriction enzyme. Both GBS methods returned considerably less SNPs than the sequence capture. Nevertheless, it was possible to distinguish between southern interior, northern interior, and coastal provenances using SNP data of the GBS experiments. GBS, especially with two restriction enzymes, seems to be a promising approach to genotype a large number of Douglas-fir trees and to obtain SNPs at low costs, which can be used in several tasks like genome-wide association studies. A large amount of sequence data and SNPs were analyzed in this thesis. Together with phenotypic information, these data will be crucial for the analysis of useful traits in Douglas-fir, like drought tolerance. Furthermore, the results concerning the Douglas-fir genome and the genetic diversity of different provenances will be beneficial in breeding programs and association studies, which in turn can be helpful to choose the optimal provenances for a given location.
Transcriptional and proteomic responses towards early nitrogen depletion in Arabidopsis thaliana
(2016) Menz, Jochen; Ludewig, Uwe
Plant roots acquire nitrogen predominantly as ammonium and nitrate, which besides serving as nutrients, also have signaling roles. Re-addition of nitrate to starved plants rapidly and di-rectly transcriptionally re-programs the metabolism and induces root architectural changes, but the earliest responses to nitrogen deprivation are unknown. In this thesis, the early transcriptional response of developed roots to nitrate or ammonium deprivation were analyzed in two Arabidopsis ecotypes contrasting in their nitrogen use efficiency: the inefficient genotype Col-0 and the efficient Tsu-0. The rapid transcriptional repression of known nitrate-induced genes proceeded the tissue NO3- concentration drop, with the transcription factor genes LBD37/38 and HRS1/HHO1 among those with earliest significant change. Some transcripts were stabilized by nitrate, but similar rapid transcriptional repression occurred in loss-of-function mutants of the nitrate response factor NLP7. In contrast, an early transcriptional response to ammonium deprivation was almost completely absent. In Col-0, the analysis was extended with the proteome and phospho-proteome resulting in a rapid and transient perturbation of the proteome induced by ammonium deprivation and a differential phosphorylation pattern in proteins involved in adjusting the pH and cation homeostasis, plasma membrane H+, NH4+, K+ and water fluxes. Fewer differential phosphorylation patterns in transporters, kinases and other proteins occurred with nitrate deprivation. The deprivation responses are not just opposite to the resupply responses, identify NO3--deprivation induced mRNA decay and signaling candidates potentially reporting the external nitrate status to the cell. Transcrip-tome comparison revealed only few N-nutrition related genes between both ecotypes contributing the increased NUE of Tsu-0, which probably relies on higher biomass accumulation. Besides, Tsu-0 confirmed the transcriptional depletion response of Col-0.
Transcriptomic analyses during infectious anemia in pigs
(2019) Mack, Sarah-Lena; Hölzle, Ludwig
Mycoplasma suis (M. suis) is a uncultivable hemotrophic bactreia parasiting red blood cells in pigs and a small range of other animals. It becomes more and more important because of leading to big economic losses in swine industry. M. suis causes anemia in pigs and is accompanied with other immunosuppressive diseases. A once infected animals is a life-long carrier and could infect other animals as well. To date, there is less information about the pathogenesis and reproduction of the bacteria and it is not possible to cultivate M. suis in vitro. One objective of the present study was to get more information about the transcriptomic changes in a pig during an infection course. Therefore, 3 splenectomized piglets were infected with the highly virulent strain KI_3806. After 2, 4 and 8 days post infection (p.i.) blood samples were taken and total RNA of blood was extracted. Microarray analyses were performed with a commercial Affymetrix array. Using microarrays more than 7000 DE genes from infected pigs could be detected. With M. suis in its host cells – the erythrocyte – we had a perfect model showing molecular interactions or signaling pathways in the M. suis infection process. With the help of the Ingenuity pathway analyses service many genes involved in immune and inflammatory response were found. Moreover typical genes involved in anemia, psoriasis and endothelial cell damage could be observed. The detection of these genes verified the depression and alteration of the immune system by M. suis resulting in evading the immune system and therefore in persisting among the organism. Another aim was to go deeper on the transcriptional level of M. suis and to get insights of the behavior of the bacteria at the time point of and after infection. RNA Sequencing was performed on a HiSeq 2000 Genome Analyzer from Illumina an resulting reads were mapped to reference sequences M. suis KI_3806 and Sus scrofa. Moreover, differential expression analysis was performed using the edgeR package. After mapping, it could be observed that on day 4 p.i. M. suis transcripts seem to be overlapped by porcine transcripts, whereas on day 8 p.i. most of the reads could be allocated to the M. suis genome resulting in almost all M. suis genes were found to be transcribed at the end of infection. When looking at the COG categories the group of proteins with “unknown function” (hypothetical proteins) represented the largest group on both days. Also a high number within the differentially expressed genes were hypothetical genes showing that these genes play an important role during infection. Further investigations are needed to confirm that the hypothetical genes also are involved in M. suis replication and recombination. In conclusion, our analysis revealed several thousand genes differentially expressed during acute IAP and numerous altered pathways and cellular processes throughout the course of host response to acute M. suis infections, thus contributing to a better understanding of the IAP pathogenesis. Moreover, this could lead to new approaches towards cultivation of M. suis as well as therapeutic treatments.
Vergleichende Transkriptomanalyse und funktionelle Untersuchungen von enterohämorrhagischen Escherichia coli nach Kultivierung in Pflanzenmedium
(2020) Bufe, Thorsten; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are human pathogens which are able to cause severe gastrointestinal diseases in humans. The gastrointestinal tract of cattle is considered as the main reservoir for EHEC and contaminated raw meat represents the primary source of infection. Yet there have been increasing reports over the last few decades of EHEC infections that were linked to the consumption of raw vegetables. Today it is generally accepted that EHEC bacteria are able to use plants as their secondary hosts, thus favouring the transmission to humans. To improve the understanding of this pathogen-plant interaction fundamental knowledge about the pathogens’ molecular adaptions towards plant material is urgently required. In the cope of this study the adaption of different EHEC strains towards components of the plant was examined. Therefore O157:H7 strain Sakai, O104:H4 strain C227-11phicu and O157:H strain 3072/96 were chosen as surrogates. In growth experiments performed with an artificial lettuce medium it could be shown that components of the lettuce were sufficient for the proliferation of the three strains. RNA-sequencing was performed to study the differential gene expression of the three strains after the growth in lettuce medium compared to the growth in M9 minimal medium. In order to compare genes according to standardized gene denotations, the differential gene expression analysis was performed on the basis of a shared genome including the genomes of the three pathogenic strains as well as the genome of Escherichia coli strain K-12 substrain MG1655. Analogous to the successful growth in presence of components of the plant an upregulation of genes involved in carbohydrate and peptide metabolism throughout all three strains was observed. Especially genes involved in the catabolism of lactose (lacZ), ribose (rbsAC) and xylose (xylF) were found to be uniformly upregulated. The greatest differences among the strains accounted for the regulation of motility and chemotaxis genes. O104:H4 strain C227-11phicu showed a strong upregulation of all three classes of the flagellar hierarchy (class I, II and III) in presence of plant derived compounds. These included genes involved in the establishment of the basal body hook structure (fli, flg), the synthesis of the flagellar filament (fliC), and the chemotaxis-system (che, tap, tar). In contrast, O157:H7 strain Sakai only featured upregulation of class I and class II genes. According to the transcriptional data both of these strains also showed increased swimming and swarming behaviour on motility plates in presence of lettuce extract. Solely O157:H- strain 3072/96, which is non-motile due to a deletion in the flhC gene, showed an upregulation of virulence factors encoded on the LEE pathogenicity island, including genes involved in the establishment of the T3SS (esc) and T3SS secreted effectors (esp). Interestingly, it was shown for O157:H- strain 3072/96 to have a powerful capacity to form biofilms in M9 minimal medium. Furthermore it was proven that the complementation of an intact flhC gene restored motility in O157:H- strain 3072/96. In this regard it could be shown that the deletion in flhC was not the mere reason for the augmented biofilm formation capacity. In addition to the biofilm formation, the strains’ potential to adhere to HT-29 cells was examined. Here a significantly increased adherence potential for O157:H- strain 3072/96 with respect to the motile strains could be observed, the lowest adherence potential was determined for O157:H7 strain Sakai. The results presented in this study clearly indicate that the different EHEC strains are capable to adapt towards the nutrient availability provided by their plantal host. It can be assumed that flagella and the chemotaxis system play a fundamental role in the finding and exploitation of the plant. Furthermore curli structures might play a crucial role in the initial adherence and the subsequent establishment of a biofilm on plant tissues. Presumably, besides the typical plant associated outbreak strain O157:H7 strain Sakai, there are further strains capable of utilizing their genetic repertoire in order to adapt towards the atypical environmental conditions within this niche. The findings of this study suggest that the strains, besides sharing multiple coinciding mechanisms, are able to adapt in a strain specific manner and use different strategies in coping with plants as their secondary hosts.