Metabolic flux analysis of the chloroplast pathway for isoprenoid biosynthesis in Arabidopsis thaliana
Final Report Abstract
Isoprenoids are a large class of natural compounds with numerous roles in plant biology as well as applications in human medicine and agriculture. This project deals with central questions in plant isoprenoid metabolism and exploits quantitative flux techniques to define the metabolic control points during their biosynthesis. The results describe the fundamental way that plants respond to environmental cues such as light and channel fixed carbon toward isoprenoid formation. Whole plant 13CO2 labeling techniques were combined with mass spectral analysis of labeled metabolites such as dimethylallyl diphosphate (DMAPP, an important isoprenoid intermediate) to calculate rates of flux in the methylerythritol phosphate (MEP) pathway of plant chloroplasts. This data is used in conjunction with enzyme activity measurements to calculate control coefficients, local indicators of metabolic control. A suite of transgenic lines expressing the gene for the first enzyme of the MEP pathway, 1-deoxyxylylose 5-phosphate synthase (DXS), was generated by screening and ranking a large number of independently transformed lines according to their DXS activities. Gradual variations in DXS activity made it possible to observe its influence on multiple downstream metabolic processes. Label introduction was accomplished with a non-invasive 13CO2 labeling technique in a dynamic labeling cuvette capable of establishing gas exchange (photosynthesis) rates prior to labeling. This labeling system provides kinetic information on the rates of carbon incorporation and partitioning into the isoprenoid pathway. We were able to demonstrate that the flux through the MEP pathway is absolutely dependent on light but that even at maximum flux only represents only about 20% of the total cellular pool of DMAPP, with the remainder likely derived from a separate pathway in the cytosol. Despite its lower standing concentration, this plastidic DMAPP pool is turned over on the scale of tens of minutes in the light, whereas its flux comes to an immediate halt when the lights are switch off. The cytosolic pathway is inactive in the light, suggesting a common motif for regulating the two isoprenoid pathways in plants. These ecophysiological experiments likewise showed that the substrates for the MEP pathway are Calvin cycle derived photosynthate, as opposed to glycolytic intermediates from the cytosol. DXS appears to be upregulated to such an extent under optimal light and temperature conditions that further upregulation of DXS activity in transgenic lines failed to produce further increases in overall pathway flux (flux control coefficient, FCC = 0), even though it had a positive effect on the concentration of DMAPP (concentration control coefficient, CCC = 0.29) and on CO2 incorporation (control coefficient = 0.135). A parallel strategy to understand the role of subcellular compartmentation in isoprenoid biosynthesis lead to the unexpected discovery that isoprenoid formation is linked to the formation and assembly of cutin monomers in the flowers of Arabidopsis. A mutant for IPP isomerase, idi2, was analyzed using metabolite profiling and was found to accumulate various dicarboxylic acids and their precursors which are normally assembled into the cutin polymer after the processing of their fatty acid precursors. The idi2 mutant produces flowers with a damaged cuticle and crinkled phenotype. Experiments to conclusively establish the molecular role of IDI2 in this process are now being completed. The information described in this project is useful for rational metabolic engineering strategies which attempt to optimize the production of isoprenoid based vitamins in crop plants, including provitamin A, vitamin E, and vitamins D and K. Future prospects of this work will focus on developing a predictive model for isoprenoid biosynthesis.
