Functional analysis of DAHP synthase isoforms in higher plants
Final Report Abstract
The 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase [EC 2.5.1.54] (DHS) is the entry enzyme of the shikimate pathway and catalyses the conversion of Phosphoenolpyruvat (PEP) and Erythrose-4-phosphate (E4P) to DAHP. To elucidate the regulation and function of the three existing isozymes of DHS in Arabidopsis and tobacco different approaches have been followed. First: All putative DHS genes of Arabidopsis and tobacco have been overexpressed heterologously in bacteria. All expressed enzymes showed DHS acitivity with comparable Km for PEP (0,11-0,17 mM) and E4P (0,24 – 0,29 mM). The isozyme DHS3 of Arabidopsis seems to have a slightly higher (50%) Vmax and a broader optimal pH range, as this isozyme still possesses 70% activity at pH 5.5 compared to the optimum at pH 7.0, while the other two isozymes just show 20-30% remaining activity. All isozymes exhibit dependency on Manganese, increased activity after reduction with DTT and further increased activity after incubation with reduced Thioredoxins. None of the isozymes was inhibited by any of the aromatic amino acids, but DHS1 was slightly activated. Second: For each isozyme, homozygous single insertion T-DNA knock-out (KO) lines of Arabidopsis plants have been generated, as well as some double KO lines. When these plants are cultivated under standard conditions they show no severe changes in phenotype compared to wild type plants, neither according shape, growth or development nor according aromatic metabolites or transcript abundance of the other DHS. Only the DHS2-KO plants displayed a slightly higher Phenylalanine concentration. But some interesting stress specific phenotypes have been demonstrated. A shift of the plants from standard to constant light conditions leads for the wild type plants to a large increase in biomass compared to plants kept under standard conditions, while all KO plants were much less affected. Further when shifted to cold treatment (10°C at day and night), the growth of most plants was strongly reduced compared to controls, but for DHS2-3-KO plants this reduction was diminished. Both effects clearly point to a central role of the DHS in plants stress response. Lacking double mutants and further RNAi plants are in progress to completely knock down all three plastidial isozymes.
Publications
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“Die drei Isoformen der DAHP-Synthase in Arabidopsis - warum sind es drei?" 22nd DBG-meeting on „Plant molecular biology“, Dabringhausen, Germany, 2009
A. Pietzke, I. Janzik
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“Comparative study of three isoenzymes of 3-Deoxy-D-arabino-heptulosonate 7- phosphate synthase in Arabidopsis thaliana”. 7th Tri-National Arabidopsis Meeting, Salzburg, Austria, 2010
A. Pietzke, A. Wiese-Klinkenberg, A. Neuwohner, S. Preiskowski, I. Janzik
