Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way.We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids, the most widespread group of pigments found in nature. Carotenoids provide distinctive coloration to flowers and fruits, important for the visual attraction of animals for pollination and seed dispersal. Carotenoids in plants are also precursors for the synthesis of the hormone abscisic acid and other apocarotenoids with biological activity [1]. They are also found in chloroplasts, where they play an essential role as accessory pigments for light