Tree roots have the potential to release carbon into deep soil layers, where this carbon is generally considered to exhibit greater stability. However, field studies that investigate the drivers of the soil organic carbon (SOC) balance in the rhizosphere of trees across soil depths and that upscale this balance to the whole soil profile are lacking. This study presents an innovative approach integrating normalized rhizosphere sampling and root density mapping to a depth of 1.5 m under trees from Mediterranean agroforestry and a tree plantation. The estimated SOC balance in the rhizosphere of the Robinia pseudoacacia trees varied from −38 kg C ha−1 to +53 kg C ha−1 at the different soil horizons, with a neutral balance at 0–0.3 m, a negative balance at 0.3–0.5 m and a positive balance at 0.5–1.0 m and 1.0–1.5 m of soil depth. When scaled up to the whole profile, the value was +50.6 kg C ha−1 for the tree plantation and +72.4 kg C ha−1 for the tree row for the agroforestry system, with no significant difference between these two estimates. The balance between hydrolytic and oxidative enzyme activities and between fungal guilds indicated increasing nutritional constraints for microbial saprotrophs at depth. In the subsoil, these nutritional constraints were locally attenuated in the rhizosphere, inducing a substantial increase in microbial abundance and triggering a pronounced shift from oligotrophic to copiotrophic communities, which in turn supported SOC enrichment. In the topsoil, the lower chemical complexity of substrates available to microorganisms increases susceptibility to saprotrophic activity, which likely underlies the observed neutral or negative SOC balances in the rhizosphere. This field study presents a scalable approach for quantifying the rhizosphere SOC balance in deep soil horizons and disentangling its biogeochemical drivers.
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