Assessing and avoiding C isotopic contamination artefacts in mesocosm-scale 13CO2/12CO2 labelling systems: from biomass components to purified carbohydrates and dark respiration

Background Quantitative understanding of plant carbon (C) metabolism by ¹³ CO ₂ / ¹² CO ₂ -labelling studies requires absence (or knowledge) of C-isotopic contamination artefacts during tracer application and sample processing. This concern has not been studied explicitly but is especially crucial in experiments at different atmospheric CO ₂ concentrations ([CO ₂ ]), when experimental protocols require frequent access to the labelling chambers. Here, we used a plant growth chamber-based ¹³ CO ₂ / ¹² CO ₂ 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 the CO ₂ supplied to them (δ ¹³ C _CO2 –43.5‰ versus –5.6‰). In this setup, dδ ¹³ C _X (the measurements-based δ ¹³ C-difference between matching samples X collected from the parallel chambers) is expected to equal dδ ¹³ C _Ref (the predictable, non-contaminated δ ¹³ C-difference ), if sample-C is completely derived from the contrasting CO ₂ sources. Accordingly, contamination ( f _contam ) was determined as f _contam = 1 – dδ ¹³ C _X /dδ ¹³ C _Ref 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 mmol mol ^-1 CO ₂ , 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 [CO ₂ ] level. [CO ₂ ] 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 mmol mol ^-1 CO ₂ , _{contamination of respiratory CO 2 was close to that of biomass- and WSC-C, suggesting it originated primarily from in vivo -contaminated respiratory substrate. Surprisingly, however, we found no evidence of contamination of respiratory CO 2 at 800 mmol mol ^-1 CO 2 . Overall, contamination likely resulted overwhelmingly from photosynthetic fixation of extraneous (contaminating) CO 2 which entered chambers primarily during (daytime) experimental activities. Conclusions The labelling facility enables months-long, quantitative ¹³ CO 2 / ¹² CO 2 -labelling of large numbers of plants with accuracy and precision across contrasts of [CO 2 ], empowering eco-physiological study of climate change scenarios. Effective protocols for contamination avoidance are discussed.}