Browsing by Subject "Matixmetalloproteinases"

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Charakterisierung der Matrixmetalloproteinasen (SlMMP1 & SlMMP2) aus Tomate (Solanum lycopersicum) und ihre Rolle in der pflanzlichen Entwicklung und Pathogeninteraktionen
(2010) Pasule, Christian; Schaller, Andreas
Matrixmetalloproteinases belong to the family of metzincins and are widely distributed in prokaryotic as well as eukaryotic organisms. Mammalian matrixmetalloproteinases function in the formation of peptide hormones, growth factors and receptor proteins and regulate important physiological processes. Little is known about the function of the related plant matrixmetalloproteinases, except for a few enzymes in plants. Their function in tomato plants is still unresolved. However, preliminary data suggest that matrixmetalloproteinases may play essential roles in the regulation of development and pathogen defense in tomato plants. The present study aimed at the elucidation of the function of the tomato matrixmetalloproteinases SlMMP1 and SlMMP2 by purification and biochemical characterization of the recombinant proteins, detailed expression analysis in tomato plants, as well as the phenotypical and molecular analysis of transgenic plants with reduced expression levels for SlMMP1/2 (SlMMP1/2-RNAi). SlMMP1/2 were expressed in E. coli and purified by affinity chromatography. Activity of recombinant MMPs was stimulated by Ca2+-ions. The highest activity was measured for SlMMP1 at pH 6,5 and for SlMMP2 at pH 7. The apparent Km-values for a fluorigenic peptide substrate were 19,5 µM for SlMMP1 and 19,9 µM for SlMMP2, with a catalytic efficiency (kcat/KM) of 0,010 and 0,024 s-1*µM-1 respectively. These data suggest redundant functions for SlMMP1 and SlMMP2. SlMMP1 was found to be expressed in all tested organs and developmental stages with highest expression levels in eight days old seedlings and stems from six weeks old plants. A constitutive expression in leaves from six weeks old plants was not observed for SlMMP2. In eight days old seedlings, SlMMP2 showed higher expression in roots and hypocotyls than in cotyledons. The tissue-specific expression of SlMMP1 and SlMMP2 suggests specific functions and substrates for the two enzymes in different tissues, inspite of their similar biochemical properties. SlMMP1/2 were localised to cell walls from hypocotyls and roots using immunohistochemistry. This confirms the assumed extracellular localisation of the enzymes. Transgenic plants with reduced SlMMP1/2 expression levels exhibit alterations in surface structure leading to necrotic lesions two weeks after germination. The lesions spread over the entire hypocotyl during later stages of development. Cellular organisation was altered in cortical tissues accompanied by cell death events in the epidermis and subepidermal cell layer. These effects spread over leaves during later stages of development. SlMMP1/2-RNAi-plants accumulate massive amounts of phenolic compounds, and exhibit impaired root development with a reduction in overall root length and reduced numbers of primary and secondary lateral roots. Many of the observed effects suggest an involvment of ethylene and a higher ethylene production was in fact confirmed in SlMMP1/2-RNAi-plants. The effects of the loss of SlMMP1/2 on gene expression were analyzd with microarrays in hypocotyls from SlMMP1/2-RNAi-plants. 522 genes were found to be differentially regulated in SlMMP1/2-RNAi-hypocotyls, with 332 up- and 190 downregulated as compared with widtype plants. Genes with roles in plant development, stress, reaction during abiotic and biotic stimuli, and genes with unknown roles in biological processes appeared to be overrepresented among the differentially regulated genes, however, statistically not significantly. Genes with functions in transport were found to be underrepresented in RNAi-plants. Based on the observation that infection with Sclerotinia sclerotiorum led to induction of SlMMP1/2 gene expression, a possible function for the enzymes in pathogen defense was analyzed. A direct involvment of the enzymes in plant defense against the fungus can be excluded, however, because no differences in fungal growth were detected between wildtype and SlMMP1/2-RNAi-plants. Furthermore, it was analyzed if there are differences in resistance against the biotrophic pathogen Xanthomonas campestris pv. vesicatoria (Xcv) between wildtype and SlMMP1/2-RNAi-plants. Bacterial growth was found to be reduced for virulent, avirulent and non-pathogenic bacteria, accompanied by strong necrosis in SlMMP1/2-RNAi-plants. These data suggest a function for SlMMP1/2 in the regulation of non-specific rections against biotic stresses. The results of the present study provide a basis for a more complete understanding of the function of SlMMP1/2 in development and pathogene defense of tomato plants.