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Background and aims Under the scenario of global change, continuous C-13-enriched CO2 labeling is a powerful tool for evaluating the interaction between plants and soil, especially the influence of elevated CO2 on the input of plant-derived C (new C) into soil in the short term. However, the methodological validity concerning the acquisition of isotopic signals and their implications in plants and soil remains ambiguous.

Methods We conducted an experiment simulating elevated CO2 with wheat planted in growth chambers. C-13-enriched CO2 with identical C-13 abundance was supplied at two CO2 concentration levels (350 versus 600 ppm) until wheat harvest. The delta C-13 values of plant tissues and soil were measured periodically during the growing season.

Results The delta C-13 values of wheat tissues under elevated (CO2)-C-13 were 15-17% higher than those under ambient (CO2)-C-13 after the heading stage, implying that the C-13 signals themselves in plants and soil were not directly representative of the influence of elevated CO2 on the C fixation in the plant and thereby the flow of plant-derived C into soil. The proportion of plant-derived C in the soil (f(new)) was calculated separately at each CO2 concentration by taking the initial soil as a reference and the corresponding delta C-13 values as parameters in the mixing model, and we found that elevated CO2 significantly enhanced the new soil C input by approximately 35.5% during our 62-day labeling. In our experiment, the f(new) value under elevated CO2 could be overestimated by up to 1.7 times if the alteration of C-13 signatures in photosynthates caused by the change in CO2 concentration is ignored.

Conclusions For methodological clarification, we suggest that 1) C-13 labeling is essential in all CO2 treatments to achieve CO2 concentration-dependent C-13 signals in plants and soil, and that 2) the influence of elevated CO2 on soil C turnover can be estimated by the difference in the f(new) under the two CO2 concentrations.