PUB - Publikationen an der Universität Bielefeld

The present work aims at understanding the regulation of the antioxidant defence system during the two most critical stages of development of oilseed plants, namely the transition from lipid based heterotrophic growth to photoautotrophic metabolism in seedlings and the development of siliques during the lipid storage phase of seeds. Special focus is given to peroxiredoxins (Prx), which are involved in detoxification of H2O2 and alkyl hydroperoxides. Arabidopsis thaliana seedlings were analyzed between 1.5 and 5 days after radicle emergence from the seed coat (DARE). They were subjected to either continuous light or light/dark-cycles, in the presence or absence of externally applied sucrose and analyzed for photosynthetic electron transport and for transcript levels of antioxidant enzymes relative to lipid mobilization, superoxide accumulation and the chlorophyll content. Chlorophyll-a fluorescence kinetics showed a transient increase in the electron transport efficiency and transient activation of non-photochemical quenching (NPQ) in seedlings grown in absence of sugar. Sugar application suppressed chlorophyll accumulation after 2.5 DARE and limited activation of NPQ. The genes for antioxidant enzymes were regulated developmentally and in a light-dependent manner. Transient induction of cytosolic Prx and of most of the enzymes of the Halliwell-Asada-cycle was observed in continuous light. In discontinuous light, nuclear genes for chloroplast antioxidant enzymes and for the mitochondrial PrxIIF responded to diurnal fluctuations and showed highest transcript levels in the light. In contrast, cytosolic PrxIID was up-regulated during the nights. Carbohydrates application affected especially APx expression and decreased APx activity, while Prx expression was hardly influenced. In general, the transcript abundance patterns showed that the chloroplast antioxidant defence system of greening seedlings is dominated by the four peroxiredoxins 2Cys-PrxA, 2Cys-PrxB, PrxIIE and PrxQ. The protein levels were already high at 1.5 DARE under all growth conditions, when APx transcript abundance and activity were low. Between 2.5 and 3 DARE, in parallel to sink-source-transition a change in the control of chloroplast antioxidant protection and metabolism took place. The antioxidant defence system shifted from a peroxiredoxin-dominated defence to the combined action of the ascorbate-dependent water-water-cycle (Halliwell-Asada-cycle) and the ascorbate-independent peroxiredoxin defence system. Leaf senescence starts with initiation of flowering. The silique wall then turns to be the main source of photoassimilates supporting seed development (Pechan and Morgan, 1985; Lewis and Thurling, 1994). Growth and photosynthesis generate ROS (Schopfer et al., 2001; Foyer and Noctor, 2000). Activation of antagonizing antioxidants prevents damage in oilseeds at the time of lipid storage. Here, Arabidopsis thaliana was used as a model oilseed plant to characterize the antioxidant systems during silique development. The information taken from the study on Arabidopsis was transferred to the oilseed crop Brassica napus. For the characterization of the antioxidant defence systems five stages of siliques development (from an early embryogenic stage to the end of maturation of seeds) were analyzed in Arabidopsis thaliana. Chlorophyll-a fluorescence parameters demonstrated high photosynthetic competence in all selected stages. Transcriptional patterns showed significant expression of chloroplastic genes and of the stress-inducible PrxIIC in young siliques compared to mature leaves. The transcript amounts of most of these genes decreased during development. The same pattern was observed for the protein levels of chloroplastic 2Cys-Prx, PrxQ and cytosolic and chloroplastic type II Prxs. The transcript levels of mitochondrial PrxIIF were constitutively high, while the protein amounts increased during silique maturation. In Brassica napus, the protein abundance pattern was similar for 2Cys-Prx, PrxQ and type II Prx. Only PrxIIF protein amounts remained constant during development. Analysis on seeds separated from the silique wall revealed expression of the type II Prxs, especially of the mitochondrial PrxIIF, in both organs. In contrast, PrxQ was only detected in silique walls, while 2Cys-Prx was found in silique walls and seeds of the middle stage in Brassica napus. It is concluded that siliques demand for strong antioxidant protection, especially in the early stages of development. The strong expression of Prx, which scavenge small alkyl hydroperoxides up to complex lipid peroxides, suggest a dominant role of these enzymes in the protection of siliques of oilseed plants.