A proteome study based on 2-D gel electrophoresis was performed in order to analyse the cold-stress response of Arabidopsis plants. The emphasis was to monitor the overall changes in the protein complement after prolonged exposure rather than short-term responses. Two different temperature regimes were used (6 degrees C and 10 degrees C) and plants were exposed to cold-stress exposure for 1 week. Protein patterns were also monitored after re-shifting plants to control conditions for a further week. To monitor gradual changes in the response to the two cold-stress conditions, the analysis was performed with DIGE technology with the inclusion of an internal standard. In the experiments using 6 degrees C, 22 spots with at least 2-fold altered expression were found; among them 18 were increased and four were decreased. When plants were exposed to 10 degrees C, 18 of these 22 spots still showed a 2-fold change; however, the alterations were, in general, more moderate than observed under 6 degrees C. Spot identification was performed by MALDI-TOF and ESI-MS/MS. Many of the proteins identified have previously been described in the context of cold-stress responses, indicating the validity of this proteome approach for further in-depth studies.
The effect of elevated CO 2 concentrations on the levels of secondary metabolites was investigated in tobacco plants grown under two nitrogen supply (5 and 8 m M NH 4 NO 3 ) and CO 2 conditions (350 and 1000 p.p.m.) each. High CO 2 resulted in a dramatic increase of phenylpropanoids in the leaves, including the major carbon-rich compound chlorogenic acid (CGA) and the coumarins scopolin and scopoletin at both nitrogen fertilizations. This was accompanied by increased PAL activity in leaves and roots, which was even higher at the lower nitrogen supply. Hardly any change was observed for the structural phenolic polymer lignin and the sesquiterpenoid capsidiol. In contrast, elevated CO 2 led to clearly decreased levels of the main nitrogen-rich constituent nicotine at the lower N-supply (5 m M NH 4 NO 3 ) but not when plants were grown at the higher N-supply (8 m M NH 4 NO 3 ). Inoculation experiments with potato virus Y (PVY) were used to evaluate possible ecological consequences of elevated CO 2 . The titre of viral coat-protein was markedly reduced in leaves under these conditions at both nitrogen levels. Since PR-gene expression and free salicylic acid (SA) levels remained unchanged at elevated CO 2 , we suggest that the accumulation of phenylpropanoids, for example, the major compound CGA and the coumarins scopolin and scopoletin may result in an earlier confinement of the virus at high CO 2 . Based on our results two final conclusions emerge. First, elevated CO 2 leads to a shift in secondary metabolite composition that is dependent on the availability of nitrogen. Second, changes in the pool of secondary metabolites have important consequences for plant-pathogen interactions as shown for PVY as a test organism.
The leaf surface of most terrestrial plants is covered with plant hairs called trichomes. These epidermal appendages are thought to contribute to many aspects of plant defense against biotic and abiotic stresses in a variety of species. Trichome development has been intensively studied in Arabidopsis, and the phytochemical composition of trichomes was analyzed in a number of plant species. However, comparatively little is known of the proteins expressed. We therefore initiated a proteome approach to better define the cellular mechanisms operating in plant trichomes using two-dimensional gel electrophoresis to separate proteins of whole leaves and isolated trichomes. Tobacco was chosen due to the presence of glandular trichomes involved in the secretion of defense compounds. Comparative image analysis of the protein patterns indicated a number of spots, which were highly enriched in trichomes relative to leaves. These spots were excised for identification by mass spectrometry. The results showed that among the proteins specifically enriched in trichomes, the components of stress defense responses were strongly represented. The high expression of stress-related proteins was verified by Western blotting. Superoxide dismutase isoforms were additionally analyzed by activity staining. Our results demonstrate feasibility of the proteome approach to elucidate the cell biology of plant trichomes.
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