Control of Substrate Supply to the Chloroplast 2-C-methyl-D-erythritol-4-phosphate Pathway

Abstract

The chloroplast 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway supplies precursors for plastidic terpenoid biosynthesis, making it a focal point for manipulating terpenoid production in plants. The MEP pathway generates the universal terpenoid intermediates isopentenyl and dimethylallyl diphosphate (IDP and DMADP) from central carbon intermediates D-glyceraldehyde 3-phosphate (GAP) and pyruvate, and the availability of these substrates is a primary determinant of its metabolic throughput in chloroplasts. While GAP is supplied via the Calvin-Benson-Bassham cycle, the origin of pyruvate is less certain. Its cryptic transport and behavior in isotopic labeling experiments has been described as the “pyruvate paradox”. Pyruvate supply in photosynthetic tissues is currently thought to depend on reimport of glycolytically derived phosphoenolpyruvate (PEP) from the cytosol, which can then undergo rapid enzymatic degradation to pyruvate. Glycolysis does not produce pyruvate directly in the chloroplast due to its downregulation in the light. Other proposed sources include direct cytosolic pyruvate import, alternative glycolytic routes such as the Entner-Doudoroff (ED) pathway, and a largely overlooked, minor activity of Rubisco, nature’s central carbon fixing enzyme. In this thesis, I demonstrate that Rubisco is in fact the primary source of pyruvate in illuminated chloroplasts. To do so, I employed a combination of biochemical, bioinformatic, physiological, and mass spectral analyses to elucidate the biogenesis and metabolism of pyruvate in chloroplasts of Arabidopsis. Using 13CO2 in vivo labeling and a low oxygen atmosphere to manipulate Rubisco activity, I confirmed its moonlighting function as the near exclusive source of pyruvate for terpenoid, fatty acid and branched-chain amino acid biosynthesis during photosynthesis. Mutant analysis and metabolic flux modeling further support this conclusion. Next, I explored the natural distribution of the ED pathway and found that, contrary to the currently accepted view, it is not a functional pathway in plants. I showed that this metabolic shunt is instead restricted to prokaryotes, most commonly proteobacteria. Crucially, it was not part of the central metabolism of the cyanobacterial ancestors of plastids, resulting in the absence of key ED pathway genes in endosymbiont genomes of plants. This conclusion partly rests on clarifying the distinct biochemical functions of two highly similar protein classes. One is present in both prokaryotes and plants (dihydroxy-acid dehydratase), while the other (ED dehydratase or EDD), is present only in prokaryotes. Their similarity led to the general assumption that the ED pathway was operational in plants. However, I show that these enzymes catalyze their respective reactions with high fidelity without overlapping substrate recognition. By cross referencing this biochemical data to phylogenetic analysis, I demonstrate that the presumed last common ancestors of plastids did not possess EDD genes needed for this pathway. Overall, this thesis clarifies our understanding of pyruvate metabolism in plants, updates our understanding of the natural distribution of the ED pathway, and connects carbon assimilation directly to terpenoid biosynthesis.