Browsing by Subject "Transkriptomanalyse"

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Bioeffector products for plant growth promotion in agriculture : modes of action and the application in the field
(2021) Weber, Nino Frederik; Neumann, Günter
Modern agriculture faces a conflict between sustainability and the demand for a higher food production. This conflict is exacerbated by climate change and its influence on vegetation, ecology and human society. To reduce land use, the reduction of yield losses and food waste is crucial. Moreover a sustainable intensification is necessary to increase yields, while at the same time input of limited resources such as drinking water or fertilizer should be kept as low as possible. This might be achieved by improving nutrient recycling and plant resistance to abiotic or biotic stress. Bioeffectors (BE) comprise seaweed or plant extracts and microbial inoculums that may stimulate plant growth by phytohormonal changes and increase plant tolerance to abiotic stress (biostimulants), solubilize or mobilize phosphorus from sparingly soluble sources such as Al/Fe or Ca-phosphates in the soil, rock phosphates, recycling fertilizer or organic phosphorus sources like phytate (biofertilizer), or improve plant resistance against pathogens by induced-systemic resistance (ISR) or antibiosis (biocontrol). For this study, in total 18 BE products were tested in germination, pot and field experiments for their potential to improve plant growth, cold stress tolerance, nutrient acquisition and yield in maize and tomato. Additionally, a gene expression analysis in maize was performed using whole transcriptome sequencing (RNA-Seq) after the application of two potential plant growth promoting rhizobacteria (PGPR), the Pseudomonas sp. strain DSMZ 13134 “Proradix” and the Bacillus amyloliquefaciens strain FZB42. Seaweed products supplemented with high amounts of the micronutrients Zn and Mn were effective in reducing detrimental cold stress reactions in maize whereas microbial products and seaweed extracts without micronutrient supplementation failed under the experimental conditions. At optimal temperature the product containing the Pseudomonas sp. strain was repeatedly able to stimulate root and shoot growth of maize plants whereas in tomato only in heat-treated soil substrate significant effects were observed. Results indicate that the efficacy of the product was mainly attributed to stimulation or shifts in the soil microbial community. Additionally, the FZB42 strain was able to stimulate root and plant growth in some experiments whereas the effects were less reproducible and more sensitive to environmental conditions. Fungal BE products were less effective in plant growth stimulation and showed detrimental effects in some experiments. Under the applied experimental conditions BE-derived plant growth stimulation mainly was attributed to biostimulation but aspects of biofertilization or biocontrol cannot be excluded, as all experiments were conducted in non-sterile soil substrates. Root and shoot growth are stimulated in response to hormonal shifts. In the gene expression analysis only weak responses to BE treatments were observed, as previously reported from other studies conducted under non-sterile conditions. Nevertheless, some plant stress responses were observed that resembled in some aspects those reported for phosphorus (P) deficiency in others those reported for ISR/SAR. Especially the activation of plant defence mechanisms, such as the production of secondary metabolites, ethylene production and reception and the expression of several classes of stress-related transcription factors, including JA-responsive JAZ genes, was observed. It also seems probable that in plants growing in PGPR-drenched soils, especially at high application rates, a sink stimulation for assimilates triggers changes in photosynthetic activity and root growth leading to an improved nutrient acquisition. Nevertheless, due to the complexity of interactions in natural soil environments as well as under practice conditions, a designation of a distinct mode of action for plant growth stimulation by microbial BEs is not realistic. A comparison of the overall results with those reported in literature or other working groups in a common research project (“Biofector”) supported the often-reported low reproducibility of plant growth promotion effects by BE products under applied conditions. Factors that influenced BE efficacy were application time and rates, temperature, soil buffer capacity, phosphorus sources and nitrogen fertilization, light conditions and the soil microbial community. Results indicate that in maize cultivation seed treatment is the most economic application technique for microbial products whereas for vegetable or high-value crops with good economic benefit soil drenching is recommended. For seaweed extracts foliar application seems to be the most economic and efficient choice. Furthermore, results emphasize the importance of a balanced natural soil microflora for plant health and yield stability.
Transcriptional profiling of Bacillus amyloliquefaciens FZB42 in response to seed and root exudates collected under different nutrient regimes
(2010) Carvalhais Costa, Lilia; von Wirén, Nicolaus
Plant growth-promoting rhizobacteria (PGPR) live in close association with plants and improve their growth. Bacillus amyloliquefaciens strain FZB42 is a prominent plant root-colonizing bacterium that is able to stimulate the growth of maize. To decipher the molecular cross-talk between B. amyloliquefaciens and crop plants, an exploratory analysis of the effect of seed and root exudates on the transcriptome of Bacillus amyloliquefaciens FZB42 was performed. Root exudates were collected from maize plants grown in an axenic hydroponic system under nutrient sufficiency or under deficient supply of nitrogen (N), phosphorus (P), iron (Fe) or potassium (K). An analysis of primary metabolites in the exudates was carried out, compared between treatments, and correlated with the transcriptional profiles of Bacillus amyloliquefaciens FZB42 that were gained after incubation of the bacterial culture with the root exudates. Higher exudation rates of citrate were found under Fe deficiency and greater release of ã-amino butyric acid under P deficiency. Based on a negative correlation observed between the average diffusion coefficient of N, P, K, and Fe in soils and the exudation rates of primary metabolites under conditions of N, P, K, or Fe deficiency, it was hypothesized that the exudation of sugars, amino acids and organic acids may reflect the availability and mobility of plant nutrients in soils. In the presence of seed and root exudates collected from nutrient-sufficient plants, genes involved in spore germination, transport and utilization of nutrients, biosynthesis pathways, multidrug transporters, motility and competence development were differentially expressed. In comparison to P, Fe and K, N-deficient maize root exudates caused a more distinguished change in the transcriptome of bacteria when they were in the logarithmic growth phase. During this growth phase, a number of genes coding for ribosomal proteins were down-regulated by N-deficient maize root exudates, indicating that bacterial activity was repressed. Exclusively in the presence of P-deficient maize root exudates, several genes associated to bacterial motility were induced. Moreover, a gene involved in the biosynthesis of the auxin precursor tryptophan was up-regulated by all deficiency treatments. In the transitional growth phase of Bacillus amyloliquefaciens FZB42, several genes were commonly down-regulated in different deficiency treatments. This finding is in agreement with previous studies showing that quorum-sensing and starvation-sensing are integrated to regulate cell entry into the transient phase. Taken together, this is the first study comparing the effect of different nutrient deficiencies on the composition of primary metabolites in root exudates of one plant species and evaluating systematically the transcriptional response of a Gram-positive PGPR to seed and root exudates collected from plants grown under different nutrient regimes. This analysis provides new information about the early communication between plant roots and PGPR and points to involved genes and processes that merit further investigation.
Transcriptomics and hormonal regulation of cluster root development in phosphate-deficient white lupin
(2014) Wang, Zhengrui; Neumann, Günter
Among crops, white lupin (Lupinus albus) represents the extraordinary ability to acquire sparingly soluble soil phosphate (Pi) by formation of cluster roots (CRs), mediating intense exudation of phosphorus (P)-mobilising root exudates (citrate, phenolics, protons and acid phosphatase). It is widely used as a model plant for investigations of P acquisition by root-induced chemical modifications of the rhizosphere. During the last two decades, a large pool of information on CR function and physiology was obtained mainly by hypothesis-driven research. Based on these findings, this study was designed to get a more comprehensive picture of the metabolic changes during CR development using a transcriptome sequencing approach. The outcome of the transcriptome analysis was the basis for the formulation of research questions on the regulation of CR formation and function to be investigated more in detail: Chapter I, focuses on transcriptome sequencing used for the first time for a systematic comparison of different stages in CR development. To get insights into the regulatory factors involved in CR formation, special emphasis was placed on hormone-related genes. Initiation of CR primordia in the pre-emergent (PE) zone was reflected by strongest expression of genes involved in transport and biosynthesis of auxins, brassinosteroids (BRs) and cytokinin receptors. Cluster root maturation, involving meristem degeneration and root hair proliferation was associated with strongly increased expression of ethylene-related transcripts and decreased expression of auxin- and BR-related genes. Also transcripts related with abscisic and jasmonic acids and cytokinin degradation were up-regulated in mature (MA) clusters. The primary metabolism, highly expressed in juvenile (JU) clusters, underwent significant modifications during CR maturation with increased contribution of Pi-independent bypass reactions, promoting biosynthesis of organic acids. Citrate catabolism and respiration were down-regulated, triggering citrate accumulation in MA clusters. Up-regulation of phenylpropanoid pathways reflected accumulation of phenolics. Increased expression of transcripts encoding ALMT and MATE transporters may be involved in the exudation of flavonoids and citrate, while up-regulation of transcripts encoding Pi transporters mediates subsequent uptake of mobilised Pi. Predominant expression of nucleotide degradation and secretory acid phosphatase in MA clusters coincides with Pi re-translocation and mobilisation of organic soil P. Up-regulation of the FIT transcription factor, usually mediating the expression of Fe deficiency responses (root hair proliferation, proton extrusion, Fe(III)-reduction, exudation of phenolics) can be observed also in MA clusters of P-deficient Lupinus albus even under Fe-sufficient conditions. This raises the question, whether FIT has a similar function in the regulation of P deficiency responses. Chapter II, addresses the question whether sucrose acts as a shoot-borne signal for CR formation. External sucrose amendments to P-sufficient plants, at concentrations similar to those in PE root zones of P-deficient plants, induced CR formation comparable to P-deficient plants. Palatinose (25 mM), and combined application of glucose/fructose (both at 12.5 mM) failed to induce CR formation under P-sufficient conditions, demonstrating a specific signal function of sucrose and excluding osmotic and carbon source effects. However, CRs induced by sucrose were not functional with respect to citrate exudation, acid phosphatase and phosphoenolpyruvate carboxylase (PEPC) activities and expression of related genes (LaMATE, LaSAP and LaPEPC) quantified by RT-qPCR. In Chapter III, the interactions of different phytohormones and sucrose on CR formation were investigated more in detail by an integrated approach of RT-qPCR, hormone translocation analyses and exogenous application of hormones or hormone antagonists. Shoot-to-root translocation of auxin was unaffected by P limitation, supporting the hypothesis that sucrose, rather than auxins, acts as major shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose or P limitation. As reported in other plant species, moderately increased production of ethylene and brassinosteroids (BRs) may induce biosynthesis and transport of root-borne auxins, indicated by increased expression of respective genes (YUCCA, PIN1, AUX1, BR, ACC_oxidase) in pre-emergent clusters. A role of BR in CR formation is further underlined by inhibitory effects of BR antagonists. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the inhibition of lateral rootlets growth during CR maturation, indicated by a massive increase of gene expression involved in ethylene production, associated with decline of transcripts with stimulatory effects (BR- and auxin-related genes). Based on these findings, models for the regulatory networks involved in CR formation (Chapter III) and function (Chapter I) have been developed.
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.