Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13CO2

Abstract

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 plant development and at sufficient levels for mass spectral analysis. We describe a system for isotopic labeling of leafy vegetables with 13C and demonstrate successful incorporation of 13C 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 13CO2 to maintain 400 μL·L−1. Nutrient solution was delivered hydroponically without allowing infusion of natural CO2 into the labeling chamber. Plants were initially grown at 22 °C ± 1 °C in constant light of 228 μmol·m−2·s−1. Upon canopy closure, anthocyanin development was promoted by reducing the nutrient solution concentration and reducing the temperature to 10.5 °C ± 1.5 °C. 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 13C incorporation into anthocyanins were evident in mass spectra of anthocyanins from labeled tissue and demonstrate successful isotopic labeling.