Non‐targeted ¹³C metabolite analysis demonstrates broad re‐orchestration of leaf metabolism when gas exchange conditions vary

Abstract

It is common practice to manipulate CO2 and O2 mole fraction during gas‐exchange experiments to suppress or exacerbate photorespiration, or simply carry out CO2 response curves. In doing so, it is implicitly assumed that metabolic pathways other than carboxylation and oxygenation are altered minimally. In the past few years, targeted metabolic analyses have shown that this assumption is incorrect, with changes in the tricarboxylic acid cycle, anaplerosis (phosphoenolpyruvate carboxylation), and nitrogen or sulphur assimilation. However, this problem has never been tackled systematically using non‐targeted analyses to embrace all possible affected metabolic pathways. Here, we exploited combined NMR, GC–MS, and LC–MS data and conducted non‐targeted analyses on sunflower leaves sampled at different O2/CO2 ratios in a gas exchange system. The statistical analysis of nearly 4,500 metabolic features not only confirms previous findings on anaplerosis or S assimilation, but also reveals significant changes in branched chain amino acids, phenylpropanoid metabolism, or adenosine turn‐over. Noteworthy, all of these pathways involve CO2 assimilation or liberation and thus affect net CO2 exchange. We conclude that manipulating CO2 and O2 mole fraction has a broad effect on metabolism, and this must be taken into account to better understand variations in carboxylation (anaplerotic fixation) or apparent day respiration.

The Impact of Photorespiratory Glycolate Oxidase Activity on Arabidopsis thaliana Leaf Soluble Amino Acid Pool Sizes during Acclimation to Low Atmospheric CO2 Concentrations

“…It is tempting to propose a stimulated shikimate pathway under low CO 2 conditions, but this would be unexpected due to the reduced photosynthetic activity producing fewer C-skeletons. A recent study found that the incorporation of photosynthetically neo-assimilated carbon towards Phe biosynthesis was reduced by up to 35% following a transfer from 380 to 140 ppm CO 2 using 13 CO 2 labeling [ 19 ]. This clearly shows a decrease in de novo biosynthesis of Phe under low CO 2 conditions and therefore intuitionally the observed increase in Phe pool size probably reflected a reduced utilization.…”

“…A recent study in Arabidopsis showed that a short-term low CO 2 treatment (4–28 h at 100 ppm) promoted the accumulation of urea cycle intermediates (including arginine and ornithine) and a differential regulation of this pathway at the transcriptional level [ 17 ]. Other studies showed that a short-term low CO 2 treatment (2 h at 140 ppm) induced a broad re-orchestration of carbon allocation to major metabolites (including glucose, organic acids and phenylalanine) in sunflowers [ 18 , 19 ].…”

13C Isotope Labelling to Follow the Flux of Photorespiratory Intermediates

“…Frozen leaf samples were extracted and analyzed as in [17]. Briefly, perchloric acid extracts were prepared in liquid nitrogen with maleate (125 µmol per sample, internal standard).…”

“…In the case of small molecules (glycine and serine), fragmentation is not feasible (glycine) or accessible by NMR (serine). Further information on exploiting fragmentation to access intramolecular 13 C contents is provided in [17,20].…”

“…The chamber can accommodate relatively large leaves and thus allows preparation of samples of sufficient size for NMR analysis. Redrawn from [17]. Simplified metabolic pathway used in calculations.…”