Isotopic labeling of plants is a powerful strategy for studying metabolic processes. Plant compounds that have incorporated isotopic labels are distinguishable from their unlabeled analogs by mass spectrometry, and therefore can be traced in complex biochemical matrices. In plant science, isotopic labeling has been used to study carbohydrate biosynthesis, nitrogen metabolism, and photosynthate partitioning (Kollman et al., 1973; Schiltz et al., 2005; Yamagata et al., 1987). Isotopic labeling of plants consumed as foods has also provided unique opportunities for understanding human nutrient metabolism (Grusak, 1997; Novotny et al., 2003).
Isotopes can be introduced into plants through roots, stems, or leaves. Root uptake of nitrogen-15 (15N)–enriched ammonium nitrate or urea has been used to characterize nitrogen distribution and remobilization in pea (Pisum sativum L.), orange (Citrus sinensis L.), and spring wheat (Triticum aestivum L.; Ma et al., 2006; Menino et al., 2007; Schiltz et al., 2005). Stem injection of isotopically labeled sulfur-35 (35S) into hard red winter wheat was used to study grain protein content (Kahlon and Chow, 1989). Isotopes may be introduced through leaves by means of photosynthetic fixation of labeled CO2. Using a pulse-chase technique in which Arabidopsis thaliana (L.) Heynh was exposed to 14CO2 for 10 min, followed by exposure to natural CO2 for another 10 min, Sun et al. (1999) measured carbon partitioning into starch and sucrose. Because of safety concerns with radioisotopes, 13CO2 is often selected for metabolic studies. Partitioning and utilization of photosynthate in soybean [Glycine max (L.) Merr.] was evaluated by periodic exposure to 13CO2 in a labeling chamber and harvests at different growth stages (Yamagata et al., 1987). Carbon losses in french bean (Phaseolus vulgaris L.) leaves during dark respiration were studied by isolating an illuminated attached leaflet in a 13CO2-enriched chamber for 10 h, followed by isolation in a respiration chamber in darkness (Nogués et al., 2004). A similar technique was developed to measure carbon fluxes in leaflets of drought-stressed tomato (Solanum lycopersicum L.; Haupt-Herting et al., 2001). Whole-plant labeling with atmospheric 13CO2 has been performed on larch (Larix Mill.) in a canopy-scale open air system over 5 d (Talhelm et al., 2007). Although this system was simpler than techniques using chambers or other enclosures, and 13C-enriched foliar respiration was detected, leaf incorporation of 13C was not significantly different from control trees.
Successful atmospheric labeling with 13CO2 poses a number of challenges. Uniform labeling is highly advantageous because the resulting labeled molecules exist as a predominant isotopomer, thus improving mass spectral detection and structure identification. To ensure uniform labeling, plants must be housed in an airtight labeling chamber, and the seal cannot be broken for irrigation. An organic growth medium may not be used because microbial metabolism of carbon substrates will dilute the 13CO2 with respired natural CO2.
In this article, we describe a study to label organic compounds in leafy vegetables with 13C by cultivation in a controlled environment containing atmospheric 13CO2 and to characterize labeled compounds by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). Due to our particular interest in anthocyanins, we have chosen red cabbage (Brassica oleracea var. capitata) as the crop for labeling, and we have used anthocyanin labeling as a primary measure of success.
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