Leaf day respiration involves multiple carbon sources and depends on previous dark metabolism.

Day respiration (R) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO during photosynthesis. R is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with R are poorly known, and this can be problematic to predict how R changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Here, we carried out a pulse-chase experiment, whereby a CO pulse in the light was followed by a chase period in darkness and then in the light. We took advantage of nontargeted, isotope-assisted metabolomics to determine non-'ordinary' metabolism, detect carbon remobilisation and compare light and dark C utilisation. We found that several concurrent metabolic pathways ('ordinary' catabolism, oxidative pentose phosphates pathway, amino acid production, nucleotide biosynthesis and secondary metabolism) took place in the light and participated in net CO efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of R, further suggesting the contribution of a variety of CO-evolving processes. Also, the cornerstone of the Krebs 'cycle', citrate, is synthetised de novo from photosynthates mostly in darkness, and remobilised or synthesised from stored material in the light. Collectively, our data provides direct evidence that leaf day respiration (i) involves several CO-producing reactions and (ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates.