While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA “cycle” does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.

Day respiration is the process by which nonphotorespiratory CO ₂ is produced by illuminated leaves. The biological function of day respiratory metabolism is a major conundrum of plant photosynthesis research: because the rate of CO ₂ evolution is partly inhibited in the light, it is viewed as either detrimental to plant carbon balance or necessary for photosynthesis operation (e.g., in providing cytoplasmic ATP for sucrose synthesis). Systematic variations in the rate of day respiration under contrasting environmental conditions have been used to elucidate the metabolic rationale of respiration in the light. Using isotopic techniques, we show that both glycolysis and the tricarboxylic acid cycle activities are inversely related to the ambient CO ₂ /O ₂ ratio: day respiratory metabolism is enhanced under high photorespiratory (low CO ₂ ) conditions. Such a relationship also correlates with the dihydroxyacetone phosphate/Glc-6-P ratio, suggesting that photosynthetic products exert a control on day respiration. Thus, day respiration is normally inhibited by phosphoryl (ATP/ADP) and reductive (NADH/NAD) poise but is up-regulated by photorespiration. Such an effect may be related to the need for NH ₂ transfers during the recovery of photorespiratory cycle intermediates.