Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase
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The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.
KeywordsArabidopsis Carotenoids Deoxyxylulose 5-phosphate reductoisomerase (DXR) Isoprenoid biosynthesis Methylerythritol 4-phosphate (MEP) pathway Taxadiene
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We thank P. Leivar and S. Sauret-Güeto for critical reading of the manuscript and E. Dellavedova, A. Orozco and Q. García for excellent technical support. We are also grateful to Zeneca Agrochemicals for the gift of CLM and the Nottingham Arabidopsis Stock Centre for providing the H2A12T7 clone and information resources. The help and advice of the staff of the Servei de Camps Experimentals (greenhouse facilities), Serveis Cientifico-Tècnics (microscopy, DNA sequencing, HPLC), and Servei d’Espectrometria de Masses (GC-MS) of the Universitat de Barcelona are greatly appreciated. The anti-DXS serum was kindly provided by Dr. P. León (Instituto de Biotecnología, Cuernavaca, Mexico). This work was supported by grants from the Spanish Ministerio de Ciencia y Tecnología and FEDER to MRC (BIO2002-1653 and BIO2005-00367) and AB (BMC2003-06833). AC, PB-P, and OB received doctoral fellowships from the Spanish Ministerio de Educación y Ciencia.
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