Radio-Metabolite Analysis of Carbon-11 Biochemical Partitioning to Non-Structural Carbohydrates for Integrated Metabolism and Transport Studies

Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 (C-11) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive C-11-based methodologies for the study of phloem transport. In this report, we present methods using C-11-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of C-11 partitioning to starch, which was previously not possible. Because of the high specific activity of C-11 and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other C-11 assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.