Discussion:
Collectively, this work identifies critical mechanisms of root and microbial control over SOM stabilization and destabilization in miscanthus systems that can promote soil C sequestration and support plant productivity. Root ingrowth did not lead to a net litter C loss (Fig. 2 ) despite increased litter decomposition due to enhanced transfer of C into more stable SOM (Fig. 3 ). Notably, we document the potential for roots to mobilize litter-derived N from POM without priming litter C loss (Fig. 2 , Fig. 4 ). We also identified that microbial nutrient or carbon limitation may alter how microbes grow and decompose litter-derived SOM, with more litter decomposition and less MAOM stabilization in organically fertilized soils (Fig. 5 ). Across all treatments, litter-derived OM was rapidly incorporated into MAOM and appeared to reflect greater microbial processing despite declining with increasing microbial biomass (Fig.6 ), supporting recent evidence that MAOM may be more dynamic than previously thought.
It appears that miscanthus roots can mine N without priming soil C losses (Fig. 2 ) and increase the C:N of litter-derived light and heavy POM (Fig. 4 ). This raises the question of how miscanthus accesses N from decomposing litter without priming C losses that are commonly observed in other ecosystems (Cheng et al., 2014; Zhu et al., 2014). One plausible mechanism may be that miscanthus roots engineer their rhizosphere microbiome composition or function to preferentially decompose N-rich litter compounds like proteins, potentially by stimulating proteolytic enzyme production (Brzostek & Finzi, 2011). While the specific mechanism remains uncertain, preferential N mining from litter has important implications for miscanthus sustainability (e.g., the propensity of miscanthus to be high yielding and build soil C). The resulting increase in remaining litter C:N may make new litter-derived SOM even more resistant to further decomposition. In addition, there has been a long-standing question of how miscanthus can maintain relatively high yields with limited N inputs (Cadoux et al., 2012). Previous research has posited that high nutrient use efficiency (Beale & Long, 1997) or the promotion of N-fixing symbionts (Davis et al., 2010) sustains N nutrition by miscanthus. Here, we show that highly effective N mining may drive the high productivity, low fertilization demand, and soil C sequestration of miscanthus cultivation (Dohleman & Long, 2009; Heaton et al., 2008; Smith et al., 2013).
Our research suggests that roots can actively support the transfer of litter derived C into more protected forms. We observed that the priming of litter decomposition from light POM was balanced by stabilization in heavy POM (Fig. 3 ). The composition of heavy POM is not as well-characterized as light POM or MAOM, but this pool is commonly assumed to be composed of stable soil macro- or micro-aggregates (Lavallee et al., 2020). Aggregate occluded SOM is largely formed through root and mycorrhizal symbiont activity (Rillig & Mummey, 2006) and often consists of partially decomposed plant and microbial organic matter fragments. This pool has a higher activation energy for decomposition than low C:N compounds like those in MAOM (Williams et al., 2018) and is more protected from decomposers than free light POM (Keiluweit et al., 2017; Kögel-Knabner et al., 2008). As such, there is an opportunity for stabilizing carbon in high C:N, heavy POM rather than lower C:N MAOM. The N requirements of low C:N SOM retention have often been cited as a criticism to efforts to use soil C management to mitigate global change (Schlesinger & Amundson, 2019). Future research efforts that investigate how roots can build new, stable, and high C:N SOM could help realize the potential of soil C sequestration to combat climate change.
Organic fertilization may promote saprotrophic soil microbes that more effectively decompose litter substrates and less efficiently form more stable heavy POM and MAOM. We found that the organic fertilizer treatments had the greatest microbial biomass and light POM decomposition, in support of our second fertilization hypothesis, but less litter C and N were incorporated into MAOM (Fig. 5 , SI Figs. 3, 4 ). On one hand, differences between fertilization treatments could arise from a shift in the microbial community structure or function with organic fertilization (Pan et al., 2014). However, other research at the site has found no significant effects of nutrient treatment on microbial diversity or mycorrhizal abundance between treatments (Kane et al. 2023, in review ). On the other hand, C vs. N limitation over microbial decomposition can regulate the rate and efficiency of SOM cycling (Averill & Waring, 2018; Schimel & Weintraub, 2003). As organic fertilization deposits both C and N, our observations could be explained by the alleviation of C limitation and induction of N limitation. In support, we observed a reduction in nitrification rates with organic fertilization relative to unfertilized plots (SI Fig. 7 ) and other research found that organic fertilization increases plot-scale microbial respiration (Kane et al., 2023, in review ). Here, microbial decomposers could increase decomposition and growth while respiring excess C and immobilizing N in living biomass rather than forming more microbially-derived MAOM (Schimel & Weintraub, 2003).
Our research supports recent theory that some component of MAOM may dynamically cycle (Kleber et al., 2021; Sokol et al., 2022). Soil C persistence theory has evolved to rely on two fundamental assumptions, that (1) microbial growth and necromass production are tightly coupled to the stabilization of microbially-derived MAOM and (2) that MAOM is physically inaccessible to decomposers, leading to slow turnover rates and long-term persistence (Cotrufo et al., 2013; Lehmann & Kleber, 2015). While our MAOM appeared to reflect greater microbial processing due to a lower, constrained C:N (Fig. 4c ), microbial biomass was weakly negatively, rather than positively, correlated with MAOM formation (Fig. 6a ). In contrast to the first assumption, this pattern supports recent findings that microbial growth and efficiency may not drive MAOM stabilization (Craig et al., 2022; Sokol et al., 2022). In contrast to the second assumption, the rapid transfer of litter carbon into MAOM (Fig. 2 ) and the loss of native MAOM over a single growing season (Fig. 6b ) suggest that some part of MAOM may turnover rapidly. Further, declining MAOM formation with microbial and root biomass (Fig. 6a , SI Fig. 5 ) support recent findings that MAOM can be continually processed by microbes (Fossum et al., 2022) and that roots can destabilize MAOM (Jilling et al., 2021; Li et al., 2021). However, other studies observe that microbial growth efficiency promotes MAOM formation (Kallenbach et al., 2016; Ridgeway et al., 2022) and that MAOM generally persists longer than POM pools (Heckman et al., 2022). As such, continuing research should be directed at determining where and under what conditions MAOM cycles dynamically.
While our experiment identified several important ways living roots and soil microbes control litter decomposition and SOM formation, some mechanisms may not have been fully captured. Our experiment was designed to separate the effects of roots vs. mycorrhizal fungi on litter C and N transformations, but our data only identifies a root effect despite the presence of mycorrhizal fungal symbionts (SI Fig. 8 ). However, the lack of differences between fungal ingrowth and total exclusion cores could be linked to the greater dependence of AM plants on root than hyphal foraging for nutrient uptake (Chen et al., 2016). Future efforts should quantify mycorrhizal fungal ingrowth to better investigate the contribution of symbiotic fungi to root-driven SOM transformations. In addition, our observations of no significant interaction between fertilization and ingrowth treatments (SI Table 1) do not support our first fertilization hypothesis that roots would have the greatest effect in unfertilized soils. However, this pattern may have been driven by the stand age of miscanthus. These plots were in the third year of growth whereas older, more nutrient limited stands exhibit greater differences in root C allocation and N acquisition (Kantola et al., 2022). As such, future efforts to investigate how nutrient availability alters living root impacts on SOM formation should leverage ecosystems with longer-term fertilization history. Despite these limitations, our data has identified several important mechanisms of SOM stabilization in situ and provides the foundation for future efforts to study how living roots and fungi alter SOM dynamics with more sophisticated measurements, under different environmental conditions, or across different ecosystems and plant-microbe interactions.
This work has expanded our mechanistic understanding of how living roots shape ecosystem processes in agricultural systems. Our finding that miscanthus roots can simultaneously prime N release from litter without an additional C release and build stable soil C has important implications for the sustainability of bioenergy production as well as the viability of restorative agricultural to offset carbon emissions. Our findings also have important implications for our understanding of the formation and stability of mineral bound carbon and nitrogen. The lack of a positive correlation between microbial and root biomass with MAOM formation, the rapid transfer of litter into MAOM, and the loss of native MAOM suggest that the mechanisms that control MAOM formation and loss are more dynamic than current theories assert (Heckman et al., 2022; Kleber et al., 2021; Sokol et al., 2022). Overall, our work suggests that living roots can mine N while stabilizing soil C. This knowledge can help improve the predictive understanding of SOM cycling that is critical to meeting the goals of restorative agriculture.