Analysis of steady state photosynthesis in alfalfa leaves

A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to (14)CO(2) under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of (14)C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles (14)CO(2) per milligram chlorophyll per hour was observed at a CO(2) level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3-phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with (14)CO(2) under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known (14)CO(2) specific radioactivity, and provision for control and adjustment of (14)CO(2) concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state.