Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Abstract

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.

Figure 1

Isolation of tomato fruit chromoplasts. Chromoplasts isolated by centrifugation in a discontinuous sucrose density gradient (15, 30, 40 and 50%) (A). Electron micrographs of chromoplasts present in the 15-30% interface (B), 30-40% interface (C) and 40-50% interface (D). Similar electron microscopy images had been taken in two independent chromoplast preparations.

Figure 2

Uptake of glucose, pyruvate and malate into isolated tomato fruit chromoplasts. Chromoplasts were incubated in the presence of the indicated concentrations of glucose (A), pyruvate (B) and malate (C) containing 3.125 Bq of [¹⁴C]-glucose, [¹⁴C]-pyruvate and [¹⁴C]-malate, respectively. Dotted lines in graphs (B) and (C) correspond to chromoplasts preloaded with Na⁺ and inorganic phosphate, respectively. Each value represents the mean and standard deviation of measures made with three separate preparations. Values are the mean +/- SE of duplicate measurements made with three independent chromoplast preparations.

Figure 3

Analysis of radiolabeled compounds in isolated tomato fruit chromoplasts. Isolated chromoplasts were incubated with [¹⁴C]-pyruvate for 24 h and then extracted with hexane:acetone:methanol (2:1:1). Labeled compounds present in the organic fraction were separated by TLC and detected by autoradiography (A). Isolated chromoplasts were incubated with [¹⁴C]-pyruvate in the absence or presence of glyceraldehyde 3-phosphate (GAP) for 24 h and extracted with hexane:acetone:methanol (2:1:1). Labeled compounds present in the organic fraction were separated by TLC and detected by autoradiography (B). Eluents for TLC were hexane:ether:acetone (60:30:20) (A) and chloroform:methanol:water (65:25:4) (B). Results displayed are representative of at least three different experiments.

Figure 4

Analysis of metabolic intermediates in isolated tomato fruit chromoplasts. Isolated chomoplasts were incubated with [¹⁴C]-pyruvate (A) and [¹⁴C]-malate (B) for 30, 60 and 120 min. At the end of the incubation periods water soluble compounds were analyzed by HPLC (Aminex HPX-87H column) coupled to a radioactivity detector. Identified compounds are indicated: 1) pyruvate, 2) acetate and 3) malate. Results displayed are representative of at least three different experiments.

Figure 5

Effect of ATP on lipid biosynthesis in isolated tomato fruit chromoplasts. Incorporation of [¹⁴C]-pyruvate into lipids was measured in the absence (white bar) or presence (grey bar) of 5 mM ATP. Values are the mean +/- SE of measurements (in duplicate) made with two independent chromoplast preparations.