Plant Methods (Aug 2025)
Assessing and avoiding C isotopic contamination artefacts in mesocosm-scale 13CO2/12CO2 labelling systems: from biomass components to purified carbohydrates and dark respiration
Abstract
Abstract Background Quantitative understanding of plant carbon (C) metabolism by 13CO2/12CO2-labelling studies requires absence (or knowledge) of C-isotopic contamination artefacts during tracer application and sample processing. Surprisingly, this concern has not been addressed systematically and comprehensively yet is especially crucial in experiments at different atmospheric CO2 concentrations ([CO2]), when experimental protocols require frequent access to the labelling chambers. Here, we used a plant growth chamber-based 13CO2/12CO2 gas exchange-facility to address this topic. The facility comprised four independent units, with two chambers routinely operated in parallel under identical conditions except for the isotopic composition of CO2 supplied to them (δ13CCO2 −43.5‰ versus −5.6‰). In this setup, dδ13CX (the measurements-based δ13C-difference between matching samples X collected from the parallel chambers) is expected to equal dδ13CRef (the predictable, non-contaminated δ13C-difference ), if sample-C is completely derived from the contrasting CO2 sources. Accordingly, contamination (f contam) was determined as f contam = 1– dδ13CX/dδ13CRef in this experimental setup. Determinations were made for biomass fractions, water-soluble carbohydrate (WSC) components and dark respiration of Lolium perenne (perennial ryegrass) stands following growth for ∼9 weeks at 200, 400 or 800 µmol mol− 1 CO2, with a terminal two weeks-long period of extensive experimental disturbance of the chambers. Results Contamination was small and similar (average 3.3% ±0.9% SD, n = 18) for shoot and root biomass and WSC fractions (fructan, sucrose, glucose, fructose) at every [CO2] level. [CO2] had no significant effect on contamination of these samples. There was no evidence for any contamination of WSC components during extraction, separation and analysis. At 200 and 400 µmol mol− 1 CO2, contamination of respiratory CO2 was close to that of biomass- and WSC-C, suggesting it originated primarily from in vivo-contaminated respiratory substrate. Surprisingly, we found no evidence of contamination of respiratory CO2 at 800 µmol mol− 1 CO2. Overall, contamination likely resulted overwhelmingly from photosynthetic fixation of extraneous contaminating CO2 which entered chambers primarily during daytime experimental activities. Conclusions The labelling facility enables months-long, quantitative 13CO2/12CO2-labelling of large numbers of plants with accuracy and precision across contrasts of [CO2], empowering eco-physiological study of climate change scenarios. Effective protocols for contamination avoidance are discussed.
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