Day respiration ( R d) is the metabolic, non-photorespiratory process by which illuminated leaves liberate CO 2 during photosynthesis. R d is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with R d are poorly known, and this can be problematic to predict how R d changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO 2 release just reflects ‘ordinary’ catabolism (glycolysis and Krebs ‘cycle’). Here, we carried out a pulse-chase experiment, whereby a 13CO 2 pulse in the light was followed by a chase period in darkness and then in the light. We took advantage of non-targeted, isotope-assisted metabolomics to determine non-‘ordinary’ metabolism, detect carbon remobilisation, and compare light and dark 13C 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 participate in net CO 2 efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of R d, further suggesting the contribution of a variety of CO 2-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 2-producing reactions and ( ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates.

Illuminated leaves assimilate CO 2 via gross photosynthesis and liberate CO 2 via photorespiration and ‘day respiration’, often denoted as R d. Day respiration is a minor CO 2-exchange component of net photosynthesis but is important for carbon use efficiency, computations of internal conductance, or the interpretation of net photosynthetic 12C/ 13C fractionation. Unfortunately, there is no simple method to measure R d and tracing the origin of C-atoms found in day-respired CO 2 is difficult. As a result, a common misconception is that day respiration is simply a catabolic, CO 2-producing flux through ordinary catabolism (glycolysis and Krebs ‘cycle’). In the past few years, considerable progress has been made in our understanding of day respiration. It appears that R d is a net flux resulting from several CO 2-generating and CO 2-fixing reactions, not only related to catabolism but also to anabolism (biosyntheses). In addition, there is now direct evidence that decarboxylating reactions are partly fed by carbon sources disconnected from current photosynthesis and this effect has consequences for isotopic mass-balance. Therefore, leaf day ‘respiration’ is much more than just CO 2 production by respiratory catabolism. Rather, it reflects whole metabolic orchestration of leaves from N fixation to secondary metabolism and it perhaps deserves another name, such as “day decarboxylations”.

Plant metabolomics has been used widely in plant physiology, in particular to analyse metabolic responses to environmental parameters. Derivatization (via trimethylsilylation-methoximation) followed by GC-MS metabolic profiling is a major technique to quantify low molecular weight, common metabolites of primary carbon, sulphur and nitrogen metabolism. There are now excellent opportunities for new generation analyses, using high resolution, exact mass GC-MS spectrometers that are progressively becoming relatively cheap. However, exact mass GC-MS analyses for routine metabolic profiling are not common, there is no dedicated available database. Also, exact mass GC-MS is usually dedicated to structural resolution of targeted secondary metabolites. Here, we present a curated database for exact mass metabolic profiling (made of 336 analytes, 1,064 characteristic exact mass fragments) focused on molecules of primary metabolism. We show advantages of exact mass analyses, in particular to resolve isotopic patterns, localise S-containing metabolites, and avoid identification errors when analytes have common nominal mass peaks in their spectrum. We provide a practical example using leaves of different Arabidopsis ecotypes and show how exact mass GC-MS analysis can be applied to plant samples and identify metabolic profiles.