Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Avda, Diagonal 643, 08028-Barcelona, Spain.
Plant Methods. 2012 Jan 13;8(1):1. doi: 10.1186/1746-4811-8-1.
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.
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.
类胡萝卜素是自然界中分布最广泛的色素群体。除了在植物生理学中的作用外，类胡萝卜素还具有营养相关性，因为它们在人类饮食中的摄入为健康带来益处。在非光合组织中，类胡萝卜素在被称为质体的专门质体中合成和储存。目前，人们对维持这些质体中类胡萝卜素生物合成高速度所需的代谢前体和辅助因子的来源知之甚少。最近的蛋白质组学数据揭示了一些可能在果实质体中起作用的生化和代谢过程。然而，考虑到叶绿体到质体的分化是果实成熟过程中非常迅速的过程，因此在蛋白质组学分析中鉴定的一些蛋白质可能代表不再具有质体功能的残留物。因此，需要进行实验验证以证明这些预测的过程实际上是否在质体中起作用。
建立了一种适用于代谢研究的高产量番茄果实质体纯化方法。放射性标记的前体在分离的质体中有效掺入并进一步代谢。分析标记的亲脂性化合物表明，质体中的脂质生物合成是一个非常有效的过程，而在类胡萝卜素中发现的相对较低的掺入水平表明，脂质生产可能代表类胡萝卜素生物合成的竞争途径。苹果酸和丙酮酸有效地转化为乙酰辅酶 A，这与质体中苹果酸酶和丙酮酸脱氢酶复合物的活性一致。我们的结果还表明，分离的质体可以在没有外源 ATP 供应的情况下主动维持合成代谢过程，因此表明这些细胞器可以以自主的方式产生这种能量辅助因子。
我们建立了一种适用于前体摄取测定和代谢分析的高产量完整番茄果实质体纯化方法。使用靶向放射性标记的前体，我们能够揭示与番茄果实质体中类胡萝卜素和脂质生物合成相关的新的生化和代谢方面。所报道的质体系统可以代表一个有价值的平台，用于解决最近转录组学和蛋白质组学数据预测的功能过程的验证和表征。
