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

BACKGROUND

Quantitative understanding of plant carbon (C) metabolism by CO/CO-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 concentrations ([CO]), when experimental protocols require frequent access to the labelling chambers. Here, we used a plant growth chamber-based CO/CO 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 supplied to them (δC -43.5‰ versus -5.6‰). In this setup, dδC (the measurements-based δC-difference between matching samples X collected from the parallel chambers) is expected to equal dδC (the predictable, non-contaminated δC-difference ), if sample-C is completely derived from the contrasting CO sources. Accordingly, contamination (f) was determined as f = 1- dδC/dδC 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 CO, 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] level. [CO] 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 CO, contamination of respiratory CO 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 at 800 µmol mol CO. Overall, contamination likely resulted overwhelmingly from photosynthetic fixation of extraneous contaminating CO which entered chambers primarily during daytime experimental activities.

CONCLUSIONS

The labelling facility enables months-long, quantitative CO/CO-labelling of large numbers of plants with accuracy and precision across contrasts of [CO], empowering eco-physiological study of climate change scenarios. Effective protocols for contamination avoidance are discussed.