Funktionelle Charakterisierung von flavinhaltigen Monooxygenasen in Arabidopsis thaliana
- Functional characterization of flavin-containing monooxygenases in Arabidopsis thaliana
Thönnessen, Alexandra; Slusarenko, Alan (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2011, 2012)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2011
Flavin-containing monoxygenases (FMOs) have been studied in animals for more than 40 years. Five known genes are decoding FMOs and the function is the detoxification of xenobiotica. The gene family of FMOs were studied for the last 10 years using the model plant Arabidopsis thaliana. FMOs contain 29 genes, classified phylogenetically into four clades. Till now the function of just three clades is known: a) clade I is involved in auxin-biosynthesis, b) clade II is involved in pathogen defense, c) clade III is involved in glucosinolate biosynthesis. Clade IV can be divided phylogeneticaly into three subclades (I-III). It contains 8 FMOs with unknown functions. The aim of this project is the functional characterization of these eight FMOs and more appropriate analysis of the FMO1-gen (clade II). For the FMO1 we just know that it is one, involved in microbial pathogen defence. The function of these FMOs was decoded by using overexpressing lines and loss of function mutants. These lines were generated, characterized and functionally analyzed. The behaviour of those FMO-genotypes (clade IV) was studied after application of different abiotic (heat/cold-stress, drought-stress, nutrient-deficient) and biotic (pathogen attack) stimuli. The results of these studies indicate that the FMO-genes (cladeIV) are not involved in the reactions of the plant to these biotic and abiotic stresses. Indeed our results show an involvement of FMO-genes in the defence against herbivorous insects (Pieris rapae). The overexpression of FMO1 (clade II) has a significant effect on the weight gain and the preference of the caterpillars. This was shown supported by feeding-assays (gain of weight) and preference-assays (two-choice test). Furthermore, a reduced expression of At1g12200 (subclade I) had a significant effect regarding the preference of the caterpillars. In addition, developmental studies demonstrated, that At1g12200 (subclade I) was involved in the early seed development. The overexpression-lines of At1g12200 showed an accelerated growth of the embryo/seedling. The concentration of the phytohormone abscicic acid (ABA), which blocks germination, was lower in comparison to the wildtype. The early germination of the overexpression-lines of At1g12200 was inhibited, due to a very low concentration of exogenous ABA, which didn’t affect the germination of the wildtype. Consequently, this functional characterization addicted till now unknown functions of the FMOs from clade IV. Some of the FMO-genes (subclade I) seemed to be involved in early seedling development in Arabidopsis thaliana and there are hints for a preference of caterpillars due to reduced expression of At1g12200. The next step of these studies would be analyzing how At1g12200 (subclade I) is involved in the biosynthesis, signal transduction and metabolism or degradation of ABA. Additionally, it should be analyzed if the preference of the caterpillars in regard to the different genotypes of At1g12200 is due to the changes of the amount of ABA. This analysis demonstrates that the FMO1-gen/protein is playing a general role in plant’s defence, not just in defence against microbial pathogens, but a very specific role for the defence against biotic stresses.