In-situ 13CO2 labeling to trace carbon fluxes in plant-soil-microorganism systems: Review and methodological guideline

Carbon (C) cycling in the atmosphere-plant-soil-microorganism system has been the focus of much recent attention, because carbon dioxide (CO2) is the most important greenhouse gas and significantly influences global climate change. It is crucial to understand the fate of newly assimilated C in plants as this process helps clarify complex underground processes, for example, the formation, decomposition, and sequestration of soil organic carbon (SOC), turnover of the microorganisms, and energy flow and matter cycling through the soil food web. Considering the cost effectiveness and feasibility of field operations, pulse labeling plants with 13C or 14C via exposure to artificially labeled 13CO2 or 14CO2 atmospheres is a more direct and efficient tool, in many cases, the only tool available to investigate C allocation to shoots, roots, and the soil and C utilization by microorganisms. Carbon allocated belowground may be (i) lost to the atmosphere by root respiration, (ii) temporarily stored in the roots, (iii) released in the form of rhizodeposition, (iv) partially stored in microbial biomass, necromass, and soil fauna, (v) respired by rhizosphere microorganisms and soil fauna, or (vi) stored for extended periods as SOC. Rhizodeposition by living roots is a vital C and energy source for rhizosphere microorganisms. Carbon flux in the rhizosphere bridges plants, soil, and microorganisms and modifies microbial SOC decomposition and, therefore, C sequestration. The objectives of this review are to (1) summarize the scope, advantages, and disadvantages of C isotope (13C and 14C) applications; (2) propose a protocol for in situ plant 13CO2 pulse labeling in which all labeling, chasing, sampling, analysis, and calculation steps are described in detail; and (3) discuss the use of combinations of 13C labeling with biomarkers such as phospholipid fatty acids (PLFAs), amino sugars, nucleic acids (DNA and RNA), and other isotope tracers such as 14C, 15N, and 33P. We believe that the review presents a reliable and standardized protocol for tracing C flow through analyzing isotope enrichment in various pools or biomarkers within atmosphere-plant-soil-microorganism systems.