Metabolic flux analysis of the chloroplast pathway for isoprenoid biosynthesis in Arabidopsis thaliana

• Applicant: Professor Dr. Michael Andrew Phillips
• Subject Area: Plant Physiology
• Term: from 2008 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 74094337

Isoprenoids are ubiquitous, structurally diverse, essential biomolecules, particularly in plants where they play diverse roles in both primary and secondary metabolism. Plant derived isoprenoids are also important in the human diet since they include several essential vitamins, such as carotene (provitamin A), α-tocopherol (vitamin E), and phylloquinone (vitamin K). Others, such as carotenoids, are noted anti-oxidant nutraceuticals, and still others are potent anti-cancer drugs. There are two independent biosynthetic routes for isoprenoid formation in plants. Unlike the cytosolic pathway present in both plants and animals, the plastid localized pathway (or methylerythritol phosphate (MEP) pathway) was only recently elucidated, and little is known about its regulation. Understanding how plastid-derived isoprenoid formation is controlled represents a central challenge in plant biology in the 21st century. To begin to quantitatively define the isoprenoid metabolic network in Arabidopsis, we propose a series of flux analysis experiments to examine the origin, control, exchange, and compartmentation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), universal isoprenoid metabolites common to both pathways. Using metabolic control analysis, flux control coefficients of select enzymes will be established by a direct kinetic approach using a suite of transgenic plant lines over- or under-expressing the gene of interest. We will focus on the first two steps in the pathway, 1- deoxyxylulose 5-phosphate synthase (DXS) and 1-deoxyxylulose 5-phosphate reductoisomerase (DXR) and use dynamic 13CO2 labeling under physiological conditions to examine flux in transgenic plant lines. Additional experiments are aimed at evaluating the role of 1-deoxyxylulose 5-phosphate (DOXP), the first committed intermediate of the MEP pathway, as an effector of flux based on measuring the elasticity of DXS and DXR toward this metabolite in planta. These findings will provide fundamental information about a central pathway in plant metabolism and thus enable manipulation of plants leading to healthier foods and greater yields of pharmaceutically important products.

• DFG Programme: Research Fellowships
• International Connection: Spain
• Host: Professor Dr. Manuel Rodríguez Concepción