Browsing by Subject "Genanalyse"

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Linking microbial abundance and function to understand nitrogen cycling in grassland soils
(2017) Regan, Kathleen Marie; Kandeler, Ellen
This thesis characterized spatial and temporal relationships of the soil microbial community, the nitrogen cycling microbial community, and a subset of the nitrogen cycling community with soil abiotic properties and plant growth stages in an unfertilized temperate grassland. Unfertilized perennial grasslands depend solely on soil-available nitrogen and in these environments nitrogen cycling is considered to be both highly efficient and tightly coupled to plant growth. Unfertilized perennial grasslands with high plant diversity, such as ours, have also been shown to have higher soil organic carbon, total nitrogen, and microbial carbon; greater food web complexity; and more complex biological communities than more intensively managed grasslands or croplands. This made the choice of study plot especially well-suited for characterizing the relationships we sought to identify, and made it possible to detect spatial and temporal patterns at a scale that has heretofore been under-examined. The first study used a combination of abiotic, plant functional group, and PLFA measurements together with spatial statistics to interpret spatial and temporal changes in the microbial community over a season. We found that its overall structure was strongly related to the abiotic environment throughout the sampling period. The strength of that relationship varied, however, indicating that it was not constant over time and that other factors also influenced microbial community composition. PLFA analysis combined with principal components analysis made it possible to discern changes in abundances and spatial distributions among Gram-positive and Gram-negative bacteria as well as saprotrophic fungi. Modeled variograms and kriged maps of the changes in distributions of exemplary lipids of both bacterial groups also showed distinct differences in their distributions on the plot, especially at stages of most rapid plant growth. Although environmental properties were identified as the main structuring agents of the microbial community, components of those environmental properties varied over the season, suggesting that plant growth stage had an indirect influence, providing evidence of the complexity and dynamic nature of the microbial community in a grassland soil. The second study took the same analytical approach, this time applying it to abundances of key members of the soil nitrogen cycling community. Marker genes for total archaea and bacteria, nitrogen fixing bacteria, ammonia oxidizing archaea and bacteria, and denitrifying bacteria were quantified by qPCR. Potential nitrification activity and denitrifying enzyme activity were also determined. We found clear seasonal changes in the patterns of abundance of the measured genes and could associate these with changes in substrate availability related to plant growth stages. Most strikingly, we saw that small and ephemeral changes in soil environmental conditions resulted in changes in these microbial communities, while at the same time, process rates of their respective potential enzyme activities remained relatively stable. This suggests both short term niche-partitioning and functional redundancy within the nitrogen cycling microbial community. The seasonal changes in abundances we observed also provided additional evidence of a dynamic relationship between microorganisms and plants, an important mechanism controlling ecosystem nitrogen cycling. The third study determined spatial and temporal interactions between AOA, AOB and NOB. These steps are related in both space and time, as the ammonia-oxidizers provide the necessary substrate for nitrite-oxidizers. Using a combination of spatial statistics and phylogenetic analysis, our data indicated seasonally varying patterns of niche differentiation between the two bacterial groups, Nitrospira and Nitrobacter in April, but more homogeneous patterns by August which may have been due to different strategies for adapting to changes in substrate concentrations resulting from competition with plants. We then asked a further question: was the microbial structure at sampling sites with high NS gene abundances fundamentally different from those with low NS gene abundances? Using a phylogenetic approach, the operational taxonomic unit composition of NS was analyzed. Community composition did not change over the first half of the season, but by the second half, the relative proportion of a particular OTU had increased significantly. This suggested an intraspecific competition within the NS and the possible importance of OTU 03 in nitrite oxidation at a specific period of time. Observed positive correlations between AOA and Nitrospira further suggested that in this unfertilized grassland plot, the nitrification process may be predominantly performed by these groups, but is restricted to a limited timeframe.
Regulation von NIMIN- und PR1-Genen aus Arabidopsis thaliana (L.) Heynh. und Nicotiana tabacum (L.) in der Salicylat-abhängigen Pathogenabwehr
(2009) Hermann, Meike; Pfitzner, Artur J. P.
Systemic acquired resistance (SAR) is an important defense mechanism of plants against a broad range of pathogens. NPR1 acts as a central regulator controlling the salicylic acid (SA)-dependent formation of SAR through interaction with TGA transcription factors leading to the induction of ?pathogenesis-related? (PR) proteins. The SA-activated expression of the PR1 genes in Arabidopsis thaliana (At) and Nicotiana tabacum (Nt) depends on cis-acting as-1-like elements with a TGACG sequence. This dissertation studies the functional relevance of NIMIN proteins and SA-dependent PR gene induction using the analysis of gene regulation. Arabidopsis has four NIMIN-genes ? N1, N2, N3 and N1b which interact independently of TGA transcription factors with NPR1. N1 and N2 have a common interaction motif and bind to the C-terminus of AtNPR1, whereas N3 binds to the N-terminus of AtNPR1. The binding site for the TGA transcription factors is located relatively central in the AtNPR1 protein. The analysis of the NIMIN gene expression in the SA-dependent signaling pathway of SAR as well as their possible involvement in the Jasmonic (JA) signaling network ought to offer new aspects for understanding the regulation of plant pathogen defense. The relevance of different as-1-like elements was studied by establishing a yeast one-hybrid system. N1b is likely to be an inactive pseudogene. Neither could transcripts be detected in untreated, SA- or JA-treated Arabidopsis plants nor was the construct N1b[GUS] with the 1135 bp 5?-region able to induce reporter gene expression in transgenic tobacco plants. Expression of N3 occurs constitutively at low levels and independently of NPR1. Treatment with SA or JA does not lead to induction of N3. Likewise, the N3 promoter is not affected by treatment with SA, JA, TMV and phytohormones. Reporter gene expression of the N3 promoter occurs constitutively in transgenic tobacco seedlings. In contrast, N1 and N2 are clearly SA-induced. After SA induction, the expression of N2 is immediate and long-lasting and regulated independently of NPR1. N1 is expressed transiently at a later point in time and is NPR1-dependent. The expression of both, N1 and N2, clearly occurs before PR1 gene expression. The analysis of GUS-reporter gene constructs confirms the early SA-dependent induction of the N1 and N2 promoters before activation of the NtPR1a promoter. Similar to the PR1a promoter, both NIMIN promoters can be induced by thiamine-HCl and show an inhibitory effect of the JA signaling network on the strength of reporter gene expression during simultaneous treatment with SA and JA. At the histological level, the N1 and N2 promoters display SA-dependent activation in leaf and root tissue of young tobacco seedlings. This activity clearly differs from the N3 promoter. The N2 promoter ? just like the AtPR1 and NtPR1a promoters ? contains an as-1-like element with a tandem repeat of TGACG, responsible for the SA sensitivity of the promoter. However the N2 as-1-like element structurally differs from the as-1-like elements in the PR1 promoters. In the N1 promoter a SA-responsive element was located in the region of -436 to -402 with respect to the translation starting point of the N1 gene. However, mutation of a TGATG repeat within this region did not result in a loss of promoter activity. Analysis of chimeric promoter constructs with foreign as-1-like elements showed that the different expression kinetics of PR1 and NIMIN genes are not encoded by the genetic information of the respective as-1-like elements. On the contrary, the as-1-like cis-acting elements affect the promoters? tissue specificity. Due to the integration of the N2-as-1-like element, the NtPR1a promoter, which is solely active in leaf tissue, adopts the typical NIMIN activity in root tissue. The presence of the 35S-as-1 element in the NtPR1a promoter leads to constitutional activation in root tissue. However, the activation in leaf tissue is still SA-dependent. In the yeast one-hybrid system, the interaction of TGA factors with as-1 and the as-1-like elements of the AtPR1, NtPR1a and N2 promoters shows only small differences in binding quality, whereas considerable differences can be detected in quantitative binding strength. Mutation of the as-1-like elements in the NtPR1a and N2 promoters results in the loss of TGA factor binding. The N1 promoter region from -436 to -399 contains a TGA binding site. Mutation of the contained TGATG repeat leads to a total loss of binding of TGA transcription factors. The neighbouring promoter context can exert both positive and negative influence on TGA factor binding. In case of the N1 promoter, the presence of adjacent promoter regions results in increased binding affinity of TGA factors. In contrast, additional NtPR1a promoter context shows a considerable reduction of TGA factor binding to as-1-like elements. Despite independent binding sites, NIMIN proteins and TGA transcription factors compete for binding of AtNPR1 in the yeast three-hybrid system. The presence of N1 and N3 thereby impedes interaction of TGA factors with NPR1, whereas simultaneous binding of N2 and TGA factor is possible without any restrictions. Binding of N1 or N2 simultaneously with N3 at the C- and N-termini of NPR1 also results in reciprocal interference suggesting a spatial folding of NPR1 where the N- and C-termini lie closely together.