In leaves, although it is accepted that CO2 evolved by dark respiration after illumination is naturally 13C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces 13C-depleted or 13C-enriched CO2. Here, we applied equations described previously for mesocosm CO2 exchange to investigate the carbon isotope composition of CO2 respired by autotrophic and heterotrophic tissues of Pelargonium×hortorum leaves, taking advantage of leaf variegation. Day-respired CO2 was slightly 13C-depleted compared to organic matter both under 21% O2 and 2% O2. Furthermore, most, if not all CO2 molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly 13C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO2 refixation and the specific nitrogen metabolism in white leaf areas.
