Anaplerotic flux into the Calvin-Benson cycle: hydrogen isotope evidence for in vivo occurrence in C-3 metabolism

As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin-Benson cycle was proposed recently. Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, C-a. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. We report deuterium fractionation signals at H-1 and H-2 of starch glucose. Below a C-a change point, these signals increase with decreasing C-a consistent with modelled fractionations by anaplerotic flux. Under standard conditions (C-a = 450 ppm corresponding to intercellular CO2 concentrations, C-i, of 328 ppm), we estimate negligible anaplerotic flux. At C-a = 180 ppm (C-i = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway. In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.