Assessing and avoiding C isotopic contamination artefacts in mesocosm-scale 13CO2/12CO2 labelling systems: from biomass components to purified carbohydrates and dark respiration
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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. Surprisingly, this concern has not been addressed systematically and comprehensively yet 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 CO 2 supplied to them (δ 13 C CO2 −43.5‰ versus −5.6‰). In this setup, d δ 13 C X (the measurements-based δ 13 C-difference between matching samples X collected from the parallel chambers) is expected to equal d δ 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– d δ 13 C X / d δ 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 µmol 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 µmol 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, we found no evidence of contamination of respiratory CO 2 at 800 µmol 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.
