Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13CO2

Isotopic labeling of plants provides a unique opportunity for understanding metabolic processes. A significant challenge of isotopic labeling during plant growth is that isotopes must be administered without disrupting plantdevelopmentandatsufficientlevelsformassspectralanalysis.We describeasystemforisotopic labelingofleafy vegetables with 13 C and demonstrate successful incorporation of 13 C into anthocyanins of preheading red cabbage (Brassica oleracea L. var.capitata L.). 'Super Red' red cabbage seedlings were grown for 34 days in an airtight acrylic labeling chamber supplied with 13 CO2 to maintain 400 mLL -1 . Nutrient solution was delivered hydroponically without allowing infusion of natural CO2 into the labeling chamber. Plants were initially grown at 22 8C ± 1 8 Ci n constant light of 228 mmolm -2 s -1 . Upon canopy closure, anthocyanin development was promoted by reducing the nutrient solution concentration and reducing the temperature to 10.5 8C ± 1.5 8C. Total shoot fresh weight (FW) was 1556 g and root FW was 491 g at harvest. Analysis of red cabbage shoot tissue by high-performance liquid chromatography/tandem mass spectrometry indicated the presence of 37 anthocyanins, of which 14 are reported here for the first time. Mass shifts representing 13 C incorporation into anthocyanins were evident in mass spectra of anthocyanins from labeled tissue and demonstrate successful isotopic labeling.