Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts
Angaman, Djédoux Maxime (Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular)
Petrizzo, Rocco (Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular)
Hernández-Gras, Francesc (Centre de Recerca en Agrigenòmica)
Romero-Segura, Carmen (Centre de Recerca en Agrigenòmica)
Pateraki, Irini (Centre de Recerca en Agrigenòmica)
Busquets Abió, Montserrat (Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular)
Boronat i Margosa, Albert (Centre de Recerca en Agrigenòmica)

Data:	2012
Resum:	Background : carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. - Results: 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. - Conclusions: 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.
Ajuts:	Ministerio de Ciencia e Innovación BIO2009-09523 Ministerio de Ciencia e Innovación CSD2007-00036 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-0026
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Carotenoid ; Chromoplast ; Tomato ; Lipid ; Ripening ; Uptake assay
Publicat a:	Plant methods, Vol. 8 (2012) , art. 1, ISSN 1746-4811

DOI: 10.1186/1746-4811-8-1
PMID: 22243738

10 p, 1.1 MB

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