Root traits and mycorrhizal fungi mediate reactive N and warming impacts on soil organic carbon

Domain: chemistry
Published: 2026-02-25
Editorial outcome: accept

Abstract
Plant roots and arbuscular mycorrhizal fungi (AMF) form a ubiquitous symbiosis in terrestrial ecosystems and critically affect soil organic carbon (SOC) dynamics. However, how roots and AMF mediate the impact of reactive nitrogen (Nr) and climate warming on SOC remains unclear. Using a multi-year Nr addition and simulated warming experiment in a semi-arid grassland, we show that Nr input and warming alter SOC by reshaping plant communities and inducing multidimensional tradeoffs among fine-root traits and AMF communities. Stable isotope (13C) tracing revealed that Nr- and warming-induced changes in roots and AMF reduced C input belowground, and mineral-associated organic C and microbial necromass in soil, while stimulating organic C decomposition. Nr input also increased soil N:P ratios and shifted AMF communities toward taxa with finer extraradical hyphae, weakening SOC protection. Together, these findings highlight root-AMF interactions as critical regulators and improve predictions of long-term SOC dynamics under future climate change.

Introduction
The net balance of soil organic carbon (SOC) accrual and loss can profoundly affect the atmospheric carbon dioxide (CO2) concentration and climate stability1,2. Over 80% of SOC originates from roots and root-associated microbes3–5, with arbuscular mycorrhizal fungi (AMF) representing an important component of these belowground inputs. Besides functioning as the primary source of plant-derived organic C entering soil, roots and AMF also promote soil C protection by facilitating soil aggregation6,7. On the other hand, they may enhance SOC loss through stimulating the growth of saprotrophic microbes and their production of exoenzymes responsible for organic C degradation7,8. The mycorrhizosphere, particularly the regions surrounding roots and AMF, is a hotspot for organic C transformations that play a crucial role in determining the fate of soil C. However, knowledge gaps in our understanding of root- and AMF-responses to climate change drivers and the resulting impact on microbial transformations of organic C critically hinder our capacity to predict and manage SOC in a changing climate.

Reactive nitrogen (Nr) input and climate warming are two major global change drivers that affect both organic C input (via plant production) to and C output (via microbial decomposition) from soil9,10. Annual global Nr input has doubled due to human activities via synthetic N fertilization, worldwide legume cultivation and industry-induced N deposition11. Simultaneously, global surface temperature has increased by 1.09°C since the Industrial Revolution, and is predicted to further increase by up to 2.6–4.8°C by the end of this century12. Because plant growth is N-limited in most terrestrial ecosystems, Nr input generally enhances plant productivity13,14 and subsequent input of organic C (e.g., litter and root exudates) to soil, favoring SOC accrual14. However, the impact of N input on organic C decomposition is less certain15–17, as increased soil N generally stimulates decomposition of relatively labile organic C15,18, while suppressing degradation of recalcitrant organic compounds such as lignin19. Consequently, N input often increases decomposition in grasslands15 but suppresses it in forests10. Warming, on the one hand, often increases organic C input to soil via enhancing plant growth, particularly in areas where water is not a limiting factor and temperature is low20. However, warming generally enhances microbial decomposition of SOC, inducing soil C losses into the atmosphere9,21. While the individual impacts of Nr input and warming on SOC dynamics have been extensively studied9,16, the roles of roots and AMF in mediating SOC responses to combined Nr and warming influence have received comparatively less attention. In particular, interactions among climate change drivers, root traits, AMF, and soil C transformations remain poorly understood, constraining our conceptual understanding of SOC stability in terrestrial ecosystems.

Root traits are key drivers of plant and ecosystem functioning that may profoundly affect SOC dynamics21–23. Plants adopt different strategies for nutrient acquisition, ranging from a “do-it-yourself” strategy with thin roots to an “outsourcing strategy” with thick roots that facilitate symbiosis with AMF24. In other words, plants can either rely on their own root system for nutrient acquisition through constructing long, narrow-diameter roots or outsource nutrient acquisition via investing in mycorrhizal associations25,26. This trade-off between investing in intrinsic root structures or in mycorrhizal partnerships constitutes a critical axis in root economics and is key to understanding root trait variation and the resulting impact on SOC22,24. However, most studies have so far examined responses of root traits and AMF communities to climate change drivers and their roles in plant resource acquisition, and the resulting impact on SOC dynamics remains largely unexplored22,27,28.

Nr input can alter traits of plant roots and their associated AMF29,30, through multiple mechanisms. First, Nr input often increases plant productivity in grassland and shifts the plant community, favoring the growth of gramineous grasses over forbs, altering C allocation belowground and/or suppressing root growth and biomass31,32. Second, Nr input increases soil N availability and alters the C economy of plants, leading to changes in root morphological traits (i.e., diameter and length) and a decrease in root C:N ratio29. Third, N-induced changes in plant C allocation may also alter the AMF community composition and external hyphal networks30, as AMF species often vary widely in their C requirements. In addition, Nr input alters soil nutrient stoichiometry, which may impact AMF community composition33,34. In phosphorus (P)-limiting soils, it would increase the soil N:P ratio, and promote plant C allocation to AMF for P exploration35.

In contrast, warming reduces soil moisture content, which can exacerbate drought stress and further constrain root proliferation in water-limited ecosystems36. While warming may enhance root cell elongation and metabolic activity37, the concurrent decline in soil moisture can counteract these benefits by limiting root expansion and function36,38. The effects of warming on AMF are similarly variable, with some studies reporting significant impacts and others finding neutral effects39,40. These inconsistencies likely arise from context-dependent factors such as different ecosystems, host plant identity, and initial AMF community composition. For example, elevated temperatures may reduce fungal hyphal growth in arid ecosystems but stimulate symbiotic efficiency in cooler regions where warming alleviates microbial metabolic constraints39,40. Yet, how the interplay between Nr input and warming affects root traits, and AMF, remains largely unexplored.

Changes in root traits and AMF communities induced by Nr input and warming are likely to cascade to SOC dynamics, influencing both organic C input and stabilization21,41. For example, Nr addition favored roots with high specific root length (SRL) and low root diameter or root tissue density (RTD)29. The resulting changes in root trait profiles are often associated with high root turnover or short lifespan42, limiting their contribution to soil aggregation and subsequent SOC formation43,44. Similarly, AMF species differ in their production of extraradical hyphae and extracellular metabolites, potentially affecting soil C formation and stabilization7,45,46. AMF communities dominated by species with extensive but finer extraradical hyphae (e.g., Paraglomeraceae and Gigasporaceae) may contribute greater C input to the soil, potentially stimulating microbial decomposition of organic matter21. In contrast, AMF communities with less extensive but thicker hyphae (e.g., Glomeraceae) may produce organic compounds that are conducive to the formation of mineral-associated organic carbon (MAOC), thereby increasing SOC stability46,47. Yet, few have explored how changes in root traits and AMF composition induced by concurring Nr input and warming may modulate soil C dynamics (both C input and output) within the mycorrhizosphere.

We initiated a long-term field study of Nr input and warming in a semi-arid grassland in 2015 to disentangle the mechanisms underpinning their impact on plant-microbial interactions, and soil C and N dynamics. To generate a comprehensive picture of changes in root-AMF traits, we quantified root production, root traits and root- or AMF-derived C, using root- and hyphae-ingrowth techniques and stable isotope tracing, and characterized AMF with the MiSeq sequencing. We hypothesized that (1) Nr input shifts plant community and alters root traits and AMF composition, increasing labile C input, stimulating microbial growth and enzyme production, and thereby stimulating soil organic C decomposition, (2) warming induces drought stress to plants, and suppresses root growth and AMF, reducing plant-derived C input to soil, and (3) over the long term, Nr and warming reduce soil C stability (i.e., MAOC and microbial necromass C).

Results
Manipulation of the Nr input and air temperature effectively modified the targeted soil physical and chemical characteristics
Field treatments of Nr input and air warming were initiated in a semi-arid grassland on China’s Yellow Loess Plateau in 2015, using experimental plots of 4 m × 4 m. Nr input (12.0 g urea-N m−2 y−1) was carried out through two equal amounts of applications, and warming was simulated via open-top chambers (OTCs; each with 60 cm top width, 75 cm bottom width, and 51.76 cm height) (Supplementary Fig. 1)48,49. Nr input did not significantly affect either air or soil temperature or soil moisture (Supplementary Tables 1 and 3). In contrast, warming significantly increased the averaged growing-season air temperature (at 20 cm above ground) by 2.0, 2.6, and 2.1 °C in 2018, 2019, and 2021, respectively (Supplementary Table 1 and Supplementary Fig. 3a–c). Warming also tended to increase soil temperature in surface layers (Supplementary Table 3), although mean soil temperature at 10 cm depth was not significantly elevated in 2018 or 2019 (Supplementary Fig. 3d, e), it increased significantly by 0.5 °C in 2021 (Supplementary Table 1 and Supplementary Fig. 3f). In comparison, warming consistently and significantly reduced mean soil moisture by 15.1, 16.8, and 13.5% in 2018, 2019, and 2021, respectively (Supplementary Table 1 and Supplementary Fig. 3g–i).

Nr input significantly increased soil available N (mainly NO3−-N), whereas warming alone had no effect on it across the 3 years (Supplementary Tables 2, 3). However, warming amplified the positive Nr effect on soil available N in 2021 (Supplementary Tables 2 and 3), likely through enhancing N mineralization, nitrification and/or reducing N losses via leaching or denitrification50. Although Nr input did not consistently affect soil available P, it consistently increased the available N:P ratio across the 3 years (Supplementary Tables 2 and 3), suggesting that high N may have induced a relative P limitation for plants and/or microbes.

Nr input and warming shifted plant community composition and root traits
To assess how Nr input and warming affect plant growth, we characterized plant community composition and measured aboveground and belowground (root) biomass in 2018, 2019, and 2021 (extensive measurements were not conducted in 2020 because the COVID pandemic limited field access). Across the 3 years, no significant interaction was detected between Nr input and warming on plant biomass (Supplementary Table 4). Nr input significantly and consistently increased aboveground net primary productivity (ANPP) by ca. 19.9 g C m−2 yr−1 (Fig. 1c and Supplementary Table 4; see also ref. 51). Concurrently, Nr input shifted plant community from forb dominance (primarily Carex spp) to grass dominance (Stipa grandis) (Fig. 1a, b), increasing the grass-to-forb biomass ratio by 290% in 2021 (Supplementary Fig. 4). In contrast, Nr input reduced mean root biomass by 26.1%, whereas warming increased it by 22.7% (both p < 0.001; Supplementary Table 4).

Fine-root growth was quantified using root-ingrowth cores to estimate fine-root production and turnover in 2018, 2019, and 2021 (Supplementary Fig. 2). Neither Nr input nor warming significantly affected root production over the 3 years (Fig. 1e). In contrast, both factors had significant but opposing main effects on root turnover with no significant interaction: Nr input increased root turnover by 54.6%, whereas warming reduced it by 30.8% (Fig. 1f and Supplementary Table 4). As for fine-root functional traits, repeated-measures analysis of variance (ANOVA) revealed no significant interaction between Nr input and warming, but significant variation among years (Supplementary Table 5 and Supplementary Figs. 5 and 6). Over the 3 years, Nr input significantly increased root N concentration by 22.0%, SRL by 17.4% and specific root surface area (SRSA) by 20.8%, but reduced root C:N ratio by 18.5% and RTD by 16.0% (Fig. 2a, b, d–f). In contrast, warming significantly reduced SRL by 25.1% and SRSA by 13.4%, but increased root diameter by 17.0% (Fig. 2c–e). These tradeoffs in root morphology reflect a fundamental shift in plant C allocation strategy mediated by changes in community composition. Specifically, Nr input promotes communities that invest in long, low-density roots, while warming favors communities that construct thicker, shorter roots.

Nr input and warming altered mycorrhizal fungal abundance and communities
To evaluate the effect of Nr input and warming, alone or in combination, on root-AMF interactions, we quantified AMF colonization of plant roots and extra-radical AMF biomass in soil. No significant interaction between Nr input and warming was detected for either parameter (Supplementary Table 5). Warming did not significantly affect AMF colonization but reduced extra-radical biomass in soil (Fig. 2g, h). In comparison, Nr input significantly reduced both parameters (Fig. 2g, h, Supplementary Fig. 7, and Supplementary Table 6), despite concurrent increases in SRL and SRSA (Fig. 2d, e). Pairwise correlations revealed that root N was significantly negatively related with RTD (Fig. 2i and Supplementary Fig. 8), reflecting a “conservation” gradient, that is, the classical fast-slow tradeoffs in root economics, where roots with low construction costs and rapid turnover contrast with more structurally dense, conservative root24,26. In addition, AMF colonization was significantly negatively related with SRL and SRSA, but positively related with root diameter and RTD (Fig. 2i and Supplementary Fig. 8). This pattern represents a “collaboration” gradient, spanning a continuum from a “do-it-yourself” nutrient acquisition strategy, characterized by thin, high-SRL roots with low mycorrhizal dependence to an “outsourcing” strategy in which plants invest in thicker roots and greater C allocation to AMF to acquire nutrients24,25. Principal component analysis (PCA) further showed that the first two components explained 72.1% of the variation in mycorrhizal colonization and root traits (Fig. 2j and Supplementary Table 10). The first axis (RTD-RNC) captures variation in RTD and N concentration, reflecting differences in root construction costs and nutrient investment strategies. The second axis (RD-AMF%-SRL) describes a gradient from thin roots with high SRL and low mycorrhizal colonization to thick roots with greater mycorrhizal dependence, corresponding to a cooperative root-AMF economic strategy24.

Alterations in soil nutrient stoichiometry and root traits under Nr input and warming may influence AMF community composition because various AMF species have different life strategies and C requirements21,30,45. We sequenced the small subunit ribosomal RNA (SSU rRNA) gene amplicons for genomic DNA extracted from both plant roots and soil in 2018, 2019, and 2021. Over the 3 years, root AMF communities were dominated by Glomeraceae (ca. 53.7%) and Paraglomeraceae (ca. 29.9%) (Fig. 3a and Supplementary Fig. 9a, c, e), but soil AMF were primarily by Glomeraceae (ca. 58.0%) and Gigasporaceae (ca. 25.9%) (Fig. 3b and Supplementary Fig. 9b, d, f). Nonmetric multidimensional scaling (NMDS) ordination and permutational multivariate analysis of variance (PERMANOVA) revealed that plant and soil AMF communities under Nr input were distinct from those under the ambient N control (Fig. 3c, d). Nr input consistently reduced Glomeraceae in roots and soils, but increased Paraglomeraceae in roots (Fig. 3a, b and Supplementary Fig. 9). In comparison, warming altered the taxonomic composition of AMF communities primarily in roots, with a significant decrease in Glomeraceae across the 3 years (Fig. 3), whereas Paraglomeraceae showed a significant increase only in 2021 (Supplementary Table 7). A significant interaction between Nr input and warming was detected solely for soil Gigasporaceae (Supplementary Table 7). Soil available N:P ratio correlated positively with abundances of Gigasporaceae and Paraglomeraceae, but negatively with Glomeraceae abundance (Supplementary Fig. 10). Compared to Glomeraceae, Gigasporaceae and Paraglomeraceae often form extensive extra-radical hyphae to facilitate plant uptake of less mobile nutrients such as P52. In P-limiting soils, plants are likely to favor AMF taxa that can effectively acquire P nutrient53. In addition, most gramineous grasses have highly branched fibrous root systems54 and are less dependent on AMF hyphae than forbs to explore and absorb nutrients from soil55. Together, altered plant community composition, accompanied by a decrease in photosynthate allocation belowground under Nr input, may promote plants to construct roots with high SRL, low root diameter and/or low RTD, while reducing AMF colonization of roots and/or favoring AMF species with lower C requirement.

Nr input and warming altered plant-derived C accumulation in soil
To determine whether changes in roots and AMF induced by Nr input and warming alter plant-derived organic C in soil, we first quantified ROOT-derived C (here ROOT refers to roots with AMF) and AMF-derived C inputs to soil, utilizing a dual ingrowth-core isotopic technique8,21. Because AMF are obligately biotrophic, three mesh sizes were used to isolate AMF effects from their host roots (see Supplementary Fig. 2). The plant community at our field site is dominated by C4 plants51,56 and the background soil exhibits a depleted 13C signature (δ13C = −23.49‰). In May 2019, ROOT- and hyphae-ingrowth cores filled with maize field soil (δ13C = −14.35‰) were installed (see Methods for more details). After retrieval in 2021, 13C enrichment in the cores was quantified to determine AMF- and ROOT-derived C inputs. Results showed that annual ROOT-derived C ranged from 79.6 to 152.5 g C m−2 after 2-yr incubation (Fig. 4b), generally exceeding aboveground litter mass C in their respective treatments (Fig. 1c). A significant interaction between Nr input and warming was detected for ROOT-derived C, but not for AMF-derived C (Fig. 4a, b and Supplementary Table 8). Both Nr input and warming independently reduced AMF-derived C inputs to soil (Fig. 4a). Relative to the control, Nr input alone, warming alone and their combination significantly reduced ROOT-derived C by 38.0, 40.3, and 41.2, respectively (Fig. 4b).

For the MAOC, no significant interaction was detected for either ROOT- or AMF-derived MAOC (Fig. 4c, d). Instead, Nr input had a significant negative main effect on both ROOT- and AMF-derived MAOC, while warming exerted a significant negative main effect only on AMF-derived MAOC (Fig. 4c, d). However, a significant interaction between Nr input and AMF presence was detected for total soil MAOC (Supplementary Fig. 11b) and Ca-associated organic C (Ca-OC) (Fig. 5a and Supplementary Table 9). Specifically, in the presence of AMF alone, Nr input significantly reduced total MAOC (Supplementary Fig. 12b) and Ca-OC (Fig. 5a). Warming also significantly decreased Ca-OC (Fig. 5a) but had no effect on Fe-associated organic C (Fe-OC) (Fig. 5b). Additionally, compared with the control, Nr input, warming and their interaction significantly reduced microbial necromass C by 11.4, 10.5, and 15.0, respectively (Fig. 5c). In contrast, the presence of AMF alone or roots with AMF had no effect on microbial necromass C (Fig. 5c).

Regression analysis further showed that AMF colonization, AMF extra-radical biomass, and Glomeraceae abundance in roots and/or soil were positively correlated with AMF-derived C and MAOC (Supplementary Figs. 12 and 13). Moreover, RTD positively correlated with ROOT-derived C and MAOC accumulation, whereas root diameter, root Paraglomeraceae, and soil Gigasporaceae abundance negatively correlated with them (Supplementary Figs. 13 and 14). Together, these results indicate that shifts in root traits and AMF community induced by Nr input and warming had affected the contribution of ROOT- and/or AMF-derived C to not only the labile C pool but also to the stable MAOC and microbial necromass C.

Nr input but not warming promoted ROOT- and AMF-mediated decomposition of SOC
We further examined how changes in roots and/or AMF under Nr input and warming affected decomposition of organic residues. ROOT- and hyphae-ingrowth cores (see Supplementary Fig. 2), each containing a mixture of 1.8 g C plant materials (13C-enriched C maize residues; δ13C = −11.58‰) and 150 g of 13C-depleted C soil (δ13C = −23.49‰), were installed in the field in September 2018. After 1-yr incubation, Nr input significantly promoted decomposition of newly added organic residues (i.e., reduced 13C remaining) in the presence of AMF alone and in ROOT-ingrowth cores (Fig. 5d). In contrast, warming had no significant effect on residue C decomposition in the presence of either live roots or AMF alone (Fig. 5d), despite increasing root biomass (Fig. 1d), likely because warming-induced water stress on soil microbes offset the accelerated effect of increased root C on microbes20,49.

Alteration in organic C decomposition under Nr input was closely related to root traits and AMF community composition. Residue-derived C remaining in the ROOT-ingrowth cores was negatively correlated with SRL and SRSA, but positively with root diameter and RTD (Supplementary Fig. 17), suggesting that thinner, longer roots enhance organic C decomposition through microbial priming21,57,58. Additionally, residue-derived C remaining in the AMF-ingrowth cores was positively correlated with AMF colonization rate (Supplementary Fig. 15) and Glomeraceae abundance (Supplementary Fig. 16a), but negatively with Paraglomeraceae abundance in plant roots (Supplementary Fig. 16b). These results suggest that increased root turnover, reduced root structural integrity, and AMF community shifts collectively enhance microbial decomposition of SOC by increasing substrate availability and reducing physiochemical protection of soil organic matter.

Nr input and warming-induced multidimensional tradeoffs among roots and AMF
Results from our multi-year study showed that Nr input and warming significantly modified root traits and shifted plant and AMF community composition, inducing multidimensional tradeoffs among roots and AMF (Fig. 6). First, Nr input often favors gramineous grass species over forbs in grasslands59,60, which were also observed in our study (Fig. 1a, b). This induced plant community alteration elicited a subsequent tradeoff between coarse and fine roots at the community level (Fig. 2). Grasses, which typically possess highly branched, fibrous root systems, differ from forbs in root dynamics and C allocation. Specifically, fibrous roots have short lifespans and exhibit faster turnover rates compared to the taproots of forbs22,54. This alteration in root architecture and turnover dynamics has implications for ROOT-derived C inputs into the soil. Second, Nr input increased root N concentration while reducing RTD (Fig. 2a, f), reflecting a root tissue conservation gradient that represents the classical tradeoff between fast and slow return for plant C investment24. Third, Nr input and warming altered root traits, such as root diameter, which directly affected mycorrhizal colonization rate (Figs. 2 and 6). For example, Nr input increased SRL and SRSA, while reducing mycorrhizal colonization of roots (Fig. 2d, e, g). This indicates that plants experiencing Nr additions may optimize their C allocation belowground for resource acquisition through increasing root length and surface area over investment in AMF24–26. Finally, Nr input and warming induced a shift in the AMF community toward Paraglomeraceae and/or Gigasporaceae with more extensive extra-radical hyphae than commonly dominant Glomeraceae species (Fig. 3a, b). This shift may represent a response mechanism through which plants adjust their photosynthate allocation in response to altered resource limitations61,62, such as reduced water and P availability. Together, the observed alterations in root traits and AMF communities under Nr input and warming directly affect the C exchange and turnover at the root-soil interface and cascade to affect soil C dynamics.

Implications for grassland SOC dynamics in a changing climate
Our study provides direct evidence illustrating that changes in root and AMF traits induced by Nr input and warming play pivotal roles in mediating soil C dynamics through multiple mechanisms (Figs. 2, 4, and 6). First, alterations in root traits and AMF communities can directly affect organic C input from plants. Root lifespan often negatively correlates with SRL and positively correlates with RTD41,42, as thinner, less dense roots with high SRL and low RTD are characterized by higher rates of mortality and turnover faster. Increased fine-root turnover under Nr input may increase ROOT-derived C input63, but decreased AMF extra-radical mycelial biomass likely reduces plant-derived C input. Second, altered C input may alter microbial decomposition of organic C. As soil saprophytic microbes are generally more C-limited rather than N and P-limited in temperate grasslands, this labile C from root turnover likely to enhance growth of saprotrophic microbes and production of exoenzymes for degrading residues, stimulating the decomposition of soil organic C64,65, as evidenced by the significant negative correlation between root turnover and residue-derived C remaining in ROOT-ingrowth cores (Supplementary Fig. 17e). Third, shifts in root traits and AMF community likely alter abiotic and biotic protection of organic C against microbial attacks66. Because plant roots and AMF hyphae are major binding agents that enmesh microaggregates into macroaggregates43,67, decreased RTD and/or AMF under Nr input (Fig. 2f–h) may reduce the formation and stability of macroaggregates, diminishing soil C protection. At the micron and molecular scales, chemical bindings between organic C compounds and reactive minerals are key in maintaining the C persistence in soil66,68. While the Ca-OC binding primarily confers soil C stability in Ca-rich calcareous soils, Fe-OC linkage is believed to be essential for C stability in acidic soils69,70. Roots with AMF reduced Fe- and Ca-OC under Nr input and warming, respectively (Fig. 5a, b), suggesting that root-AMF associations may promote the turnover of mineral-associated C and reduce the stability of SOC under future Nr input and warming scenarios. Finally, AMF species differ in their capacity to generate secondary metabolites that contribute to the MAOC pool46,47. Glomeraceae species, but not Paraglomeraceae or Gigasporaceae species, are believed to produce glycoprotein glomalins46, which function as a strong binding agent for microaggregates71. Additionally, Glomeraceae species are often associated with faster growth rate and higher turnover, leading to a great contribution of their necromass to the MAOC pool27. The positive correlations between Glomeraceae abundance and AMF-derived C or MAOC, but the negative correlations between Paraglomeraceae and/or Gigasporaceae abundance and AMF-derived C or MAOC (Supplementary Fig. 13), provide indirect evidence that different AMF lineages have distinct functional impacts on soil C dynamics, and that the observed community shift toward taxa with finer and less dense hyphae may reduce AMF capacity in enmeshing soil aggregates and protecting organic C.

Taken together, our results demonstrate that Nr input and warming induce multidimensional tradeoffs among different plant species, roots and AMF, and/or different AMF species (Fig. 6). These tradeoffs enhance root and AMF turnovers and C availability for saprophytic microbes, but may reduce root- and AMF-mediated protection of soil organic compounds. This could promote SOC turnover and thus compromise the potential of soil C sequestration. These findings offer insights into plausible mechanisms underlying the impact of reactive N and warming on plant-microbial interactions and SOC dynamics, which is necessary for improving predictions of soil C balance and terrestrial C-climate feedbacks under future climate change scenarios.

Methods
The study site and the field experimental design
The study was conducted in a grassland on the Loess Plateau at the Yunwu Mountains Natural Preserve (36°13′−36°19′ N, 106°24′−106°28′ E; ~2000 m a.s.l), northeast of Guyuan City, Ningxia Province, China. The site has a semi-arid climate with a mean annual precipitation (MAP) of ~455 mm, 60–70% of which occurs during the plant growing season (June to September). The mean annual air temperature is 6–7 °C, with the coldest in January (ca. −7 °C) and the hottest in July (ca. 23 °C). The soil is classified as a montane gray-cinnamon, Calci-Orthic Aridisol (Chinese classification) or Haplic Calcisol (FAO classification). The topsoil layer (0–10 cm) contained 40.2 g C and 4.0 g N kg−1 soil, and had a pH of ca. 8.048,49. The grassland is dominated by Stipa grandis, Stipa przewalskyi, Carex spp, Artemisia sacrorum, Chrysanthemum lavandulifolium, and Saussurea alata51,56. Prior to 1986, the grassland was intensively managed, and farmers harvested all the aboveground biomass for fuel or animal forage over nearly 30 years. This long-term removal prevented the accumulation of plant residues on the soil surface.

A long-term climate manipulation experiment was initiated in June 2015 on a largely flat hilltop (Supplementary Fig. 1). The experiment employed a full-factorial randomized complete block design with two treatment factors: N input (0 vs. 12 g N m−2 yr−1) and temperature (ambient vs. warming). The four different treatments were then formed: (a) ambient temperature and no N addition (Control, Con), (b) N addition (N), (c) warming (W), and (d) combined N addition and warming (N+W). Each of the four replicate blocks contained four plots (each at 4 m × 4 m), separated by a 1.5-m buffer strip. Reactive N (12.0 g N m−2 yr−1) was added as commercial, granular urea dissolved in water twice annually (6.0 g N m−2 in mid-May and late-June) to simulate fertilization practices, which is common in many areas of the Loess Plateau to increase the yield of forage grass60,72. The N rate was chosen based on our previous study60 showing that high N input but not low N input significantly altered plant community composition. The warming treatment was achieved using OTCs (Supplementary Fig. 1) because electricity is not accessible in the remote Preserve. A shaded thermocouple and a portable temperature meter were used manually to measure air temperature (15 cm above the ground) and soil temperature (10 cm depth) during the growing seasons of 2018 and 2019. In 2021, air and soil temperatures were continuously monitored using the S20A5 probe (Xuzhou Fala Co., Ltd., Xuzhou, China). Soil moisture was measured using a TDR-100 probe (Spectrum Technologies Inc., Plainfield, IL, USA) during the growing seasons of 2018, 2019, and 2021.

Plant and soil sampling
Plant aboveground biomass and community composition were collected in mid-August 2018, 2019, and 202151,73. Two randomly placed 0.5 × 0.5 m quadrats per plot were harvested, and all living vascular plants were sorted to species, oven dried at 65 °C for 48 h, and weighed. Plants were classified into three functional groups: gramineous grasses, forbs and subshrubs. After biomass harvesting, three soil cores (5 cm in diameter, 0–10 cm depth) were collected and composited into one sample per plot. All the composite samples were stored in a cool box and transported by express mails within 2–4 days to the laboratory in Nanjing for processing. Aboveground biomass in all plots was clipped annually in early September after soil sampling, and clippings were removed from each plot to simulate animal grazing.

Soil samples were sieved (2 mm) to remove rocks and collect roots. Subsamples of soil and roots were immediately frozen at −20 °C for molecular analysis, while the remainder was kept at 4 °C for chemical and microbial analyses. All the microbial analyses were either completed or initiated within 2 to 4 weeks after the sieving. Roots separated from the soil were thoroughly washed, dried at 65 °C, and weighed to determine biomass. Root C and N were determined using an elemental analyzer (Vario micro cube, Elementar, Germany).

Analyses of soil chemical properties
Soil inorganic-N (NH4+-N and NO3−-N) and dissolved organic carbon (DOC) were extracted from 20 g (dry soil equivalent) of soil using 0.5 M K2SO4. Inorganic-N and DOC concentrations were quantified with a flow injection auto analyzer (Skalar SAN++, Skalar Inc., Breda, The Netherlands) and a total organic C analyzer (Vario TOC cube, Elementar, Germany), respectively. Soil available P was determined using the molybdate blue colorimetric method following the extraction with 0.5 M NaHCO374.

Determination of root production, root turnover, and root traits
To assess how Nr input and warming affect fine-root dynamics, we employed ROOT ingrowth cores (10 cm tall × 5 cm i.d.; see Supplementary Fig. 2 for details) to determine root production, turnover and traits. Cores were filled with root-free field soil and installed in May 2018, September 2018, and September 2019. Soils in the ingrowth cores were collected from the study area (0–10 cm depth), sieved through a 2 mm mesh to remove roots and stones, and compacted to match the original soil bulk density. A total of 200 g of soil (dry weight equivalent) was added to each core. Cores were installed in May 2018, September 2018, and September 2019, and retrieved in September 2018, 2019, and 2021, respectively. Due to the COVID-19 pandemic, cores installed in 2019 were retrieved in 2021. Newly grown roots were carefully separated from soil, washed with tap water and analyzed for root traits (see below for details). Roots samples were oven dried at 65 °C and weighed as dry mass (M) to determine fine root production73. The fine root turnover from 2018 to 2021 were then calculated using the following equation73,75:

Fine root turnover (yr−1) = Annual belowground production / Maxium standing biomass (1)

Each fresh root sample was scanned using a scanner (Epson Perfection v850, Suwa, Japan), and root morphological parameters (i.e., average diameter, root length (L), surface area (A), and volume (V)) were analyzed using WinRHIZO (Regent Instruments Inc., Quebec, Canada). SRL, SRSA and RTD of fine roots were then calculated as ratios of root total length to dry mass (L/M), root surface area to dry mass (A/M), and fine root dry mass to its volume (M/V), respectively.

Analyses of AMF in roots and soil
AMF colonization of roots was quantified using the gridline-intersect method76. Briefly, dried roots from each plot were cut into ~1 cm segments, and ~100 segments were randomly chosen and rehydrated in deionized water for ca. 10 h. The rehydrated root samples were then cleared in 10% (w/v) KOH at 90 °C for 30 min, and acidified in 1% (v/v) HCl for 1 h. The root segments were subsequently stained in an acidic glycerol-trypan blue solution (50% glycerol (v/v), 1% hydrochloric acid (v/v), and 0.05% trypan blue (w/v)) and heated for 30 min in a steamer. Stained roots were fully washed with water and spread on a Petri dish with grid lines, and examined for AMF colonization under ×40 magnification by counting ca. 100 intersects per sample, using a stereomicroscope (SZN71, Sunny Optical Technology Co., Ltd, China).

AM fungal biomass in soil was measured using the phospholipid fatty acid (PLFA) method77. Briefly, freeze-dried soil samples (5.0 g) were weighed to extract total lipids in a chloroform-methanol-citrate buffer (1:2:0.8, v/v/v) after shaking for 1 h. The extracted lipids were fractionated into neutral, glyco- and phospholipids on a silicic-acid column by eluting chloroform, acetone, and methanol, respectively. The separated fatty acid methyl esters were analyzed by a gas chromatograph (GC) (7890A, Agilent Technologies, Santa Clara, CA, USA) coupled with a MIDI peak-identification system (MIDI, Inc., Newark, DE, USA). It is worth mentioning that the PLFA 16:1ω5 biomarker can also be present in Gram-negative bacteria78. However, since the neutral lipid fatty acid (NLFA)/PLFA 16:1ω5 ratio measured in the present study was larger than one (see Supplementary Methods and Supplementary Fig. 7g, h for details), we consider the PLFA 16:1ω5 to be a good proxy for AMF biomass in this study.

Composition of AMF communities in roots and soil were analyzed through Illumina MiSeq sequencing of the SSU rRNA gene in the Glomeromycota79,80. DNA was extracted from 0.05 g of frozen root and 0.3 g of soil using FastDNA Spin and PowerSoil DNA Isolation Kits, respectively (MoBio Laboratories Inc, CA, USA), following the manufacturer’s procedure. A two-step PCR amplification of all root and soil DNA extracts was performed using primer sets of GeoA2/AML2 (first round; CCAGTAGTCATATGCTTGTCTC/GAACCCAAACACTTTGGTTTCC) and NS31F/AMDGR (second round; TTGGAGGGCAAGTCTGGTGCC/CCCAACTATCCCTATTAATCAT). In the first PCR reaction, primers were connected to partial MiSeq sequencing adapters and barcodes. In the second one, the full adapters served as primers. Each 25 µL PCR reaction was performed with 12.5 µL 2× Taq Plus Master Mix, 7.5 µL ddH2O, 1 µL per primer, and 2 µL DNA template, under the following conditions: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min; final extension at 72 °C for 7 min. In the second reaction, diluted product (10×) of the first PCR reaction served as the template DNA. The PCR products were separated by 1.5% agarose gel electrophoresis, and purified with an Agencourt AMPure XP Kit (Beckman Coulter, Inc., USA). DNA concentration was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purified PCR products were then normalized to equimolar amounts, and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) at Allwegene Technology Co., Ltd. (Beijing, China).

Quantitative Insights Into Microbial Ecology 2 (QIIME2) toolkit was used to process raw high-throughput sequencing data81. Briefly, the “demux” plugin was used to demultiplex the raw sequences based on their unique barcodes of each sample. Primers were then trimmed from the raw reads using Cutadapt v. 1.1082. Low-quality reads (the quality score below 30, fewer than 200 bp in length, and/or containing ambiguous nucleotides) were filtered and removed. Potential chimeric reads were also detected and removed using Vsearch v. 2.7.083. The remaining sequences were clustered into different operational taxonomic units (OTUs) at a 97% identity cutoff using the UPARSE algorithm84. Finally, the MaarjAM database (http://www.maarjam.botany.ut.ee)85 of published Glomeromycotina sequences was used to assign taxonomic information. Each AMF sequence was grouped into a corresponding virtual taxon (VT) based on the following parameters: the sequence identity (≥97%), query coverage (≥97%), e-value (<1e-50) and BLAST bit score (>200)85. Each sample was rarefied to the lowest number of sequences using R package “vegan” with “rarefy” function86 to standardize sampling effort.

Effects of roots and AMF on soil C inputs and stability, and organic C decomposition
To assess the effects of roots and AMF on soil C dynamics, we quantified ROOT- and AMF-derived C inputs to soil, evaluated soil C stability by measuring MAOC, mineral-associated C fractions, and microbial necromass C, and assessed residue organic C decomposition using ingrowth-cores with three different mesh sizes (Supplementary Fig. 2).

Determination of ROOT- and AMF-derived C to soil. To quantify the contributions of plant roots and associated AMF to soil C inputs, we employed a dual ingrowth-core isotopic technique63,87, taking advantage of the difference in 13C signature between C3 and C4 plants. Because AMF are obligate biotrophs, we used three types of ingrowth cores to isolate the effects of roots and AMF. Three different mesh sizes were used to either completely exclude AMF hyphae (no-hyphae core, 0.45 µm pore size), allow AMF hyphal penetration (hyphae-ingrowth core, 25 µm pore size), or permit penetration of both AMF and fine roots (ROOT-ingrowth core, 1.8 mm pore size) (Supplementary Fig. 2). At our field site, where the plant community is predominantly C3 plants with a depleted δ13C signature of ca. −27.6‰, the cores were filled with 200.0 g of a C4 soil (δ13C = −14.35‰) and installed in May 2019. The C4 soil, obtained from a long-term corn field nearby, had a similar texture to our field soil. Due to the COVID-19 pandemic, the cores remained in situ for 2 years before retrieval in 2021. Subsamples from the extracted ingrowth cores were finely ground and analyzed for total C and δ13C using an isotope-ratio mass spectrometer with an elemental analyzer (Delta V Advantage, Thermo Fisher Scientific, Bremen, Germany) at Nanjing Agricultural University, China. The corn soil’s enriched 13C signature allowed us to calculate the proportion of ROOT- and AMF-derived C (fR) using a two-end member mixing model:

fR = (δ13Cingrowth − δ13Ccontrol) / (δ13Cmycelium − δ13Ccontrol) (2)

where δ13Cingrowth is the δ13C of soil from individual ingrowth cores, δ13Ccontrol is the δ13C from no-AMF control cores, and δ13Cmycelium/root is the δ13C of roots from each plot. We assumed that the δ13C of AMF mycelium equals that of roots based on other studies63,88. The net ROOT and/or AMF-derived C inputs (Crd-net; g C m−2) into surface soils (0–10 cm) were then calculated as:

Crd-net = p × [C] × fR × 100,000 (3)

where p represents the initial soil bulk density at 1.20 g cm−3, [C] is the soil C concentration (%) after incubation, 100,000 is the conversion factor to transform % C to g C m−2 at 0–10 cm depth.

We acknowledge that some DOC may have entered the AMF- and/or ROOT-ingrowth cores via lateral water movement, which might lead to overestimates of ROOT-derived C. However, this effect was likely minimal, because lateral flows of DOC are typically minor relative to mycorrhizal/root C fluxes, as demonstrated by numerous previous studies63,87. Also, lateral water movement in our semi-arid system is very low. In addition, we determined DOC in each ingrowth-core and found that neither Nr nor warming had significant effects on soil DOC (see Supplementary Fig. 11a), assuming that the C source of the ingrowth cores was mainly from direct plant root and their associated AMF inputs.

Density fractionation. To determine how Nr input and warming affected the contribution of roots and AMF to soil C stabilization, we separated the bulk soil from each individual ingrowth core (after 2-year incubation) into two SOC fractions: a free light fraction (F-LF) and a heavy fraction (HF, >1.8 g cm−3), following a modified density fractionation technique70. In brief, each soil sample (10 g dry weight equivalent) was oven dried at 65 °C for 72 h and dispersed in 60 mL of sodium iodide (NaI, 1.8 g cm−3) solution with beads on a rotary shaker at 180 rpm for 24 h. After dispersion, soil samples were rinsed onto a 53-µm sieve, and the fine fraction passing through the sieve (<53 µm) was collected as mineral-associated organic matter (MAOM). The MAOM fraction was oven dried at 65 °C, weighed, ground to a fine powder, and analyzed for C concentration and δ13C using an isotope-ratio mass spectrometer equipped with an elemental analyzer (Delta V Advantage, Thermo Fisher Scientific, Bremen, Germany) at Nanjing Agricultural University. ROOT- and AMF-derived MAOM-C (MAOC) was then calculated as:

MAOC = (MAOM-C (mg) / Bulk soil (g)) × fR (4)

Ca-associated C and Fe-associated C. To quantify how Nr input and warming affected the contribution of roots and AMF to soil C stabilization via Ca and Fe mineral associations, we determined Ca-associated organic C (Ca-OC) and Fe-associated organic C (Fe-OC) using a modified sequential extraction method70. Briefly, 0.25 g of the heavy fraction soil samples collected above were first extracted with 40 mL of 0.5 M sodium sulfate (Na2SO4) for 2 h. Organic C released during Na2SO4 extraction was considered as Ca-OC. Then, the remaining residue was extracted with dithionite-citrate-bicarbonate (DCB, containing 0.27 M trisodium citrate, 0.11 M sodium bicarbonate and 0.1 M sodium dithionite) at a 1:50 dry soil-to-solution ratio. The suspension was vortexed for 1 min, shaken for 16 h, and centrifuged at 3000 rpm for 10 min. Dissolved organic C in the supernatant suspension was measured as Fe-OC. Several blanks only containing DCB and deionized water were determined to correct C concentrations in the samples to eliminate the influence of C in DCB.

Microbial necromass C. To assess the influence of roots and AMF on microbial necromass within the SOC pool, we analyzed amino sugars as biomarkers of microbial residues. Soil samples from each ingrowth core were air-dried and sieved (<0.25 mm). Amino sugars (glucosamine, galactosamine, mannosamine, and muramic acid) were extracted and quantified following the standard protocol by ref. 89. Briefly, samples were hydrolyzed with 6 M HCl at 105 °C for 8 h, with myo-inositol added as an internal standard. The organic phase was evaporated at 45 °C with N2, and then resuspended in a 1:1 ethyl acetate-hexane solution. The amino sugars were separated and quantified using a GC (7890A, Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5 column and a flame ionization detector.

For the estimation of microbial necromass C, glucosamine (GluN) and muramic acid (MurN) were used as specific biomarkers for fungal and bacterial residues, respectively. Bacterial and fungal necromass C were calculated as follows:

Bacterial necromass C = MurN × 45 (5)

Fungal necromass C = (GluN / 179.17 − MurN / 251.23) × 2 × 179.17 × 9 (6)

where 45 is the conversion value from muramic acid to bacterial necromass C, 9 represents the factor converting fungal glucosamine to the fungal necromass C, and 179.17 and 251.2 are the molecular weights of GluN and MurN, respectively. The total microbial necromass C refers to the sum of bacterial and fungal necromass C.

Effects of ROOT and AMF on organic C decomposition. To assess the influence of roots and AMF on residue decomposition in the mycorrhizosphere, ingrowth cores were prepared in September 2018. Briefly, we placed 1.80 g of ca. 1 cm length dried maize litter (δ13C = −11.58‰) into each core through mixing with 150.0 g of field soil (δ13C = −23.49‰) following the method in our previous publications8,21. After 1-year field incubation (retrieved in September 2019), cores were collected and transported to the lab for processing.

To estimate the effect of roots and AMF on the losses of residue-derived C, we determined the mean proportion (fR) of the residue C in the incubated samples using the equation:

fR = (δ13CM − δ13CS) / (δ13CR − δ13CS) (7)

where δ13CM, δ13CS, and δ13CR present the mean isotopic signature for the mixture M (incubated samples of soil/residue mixture), source S (soil, −23.49‰) and source R (residue, −11.58‰), respectively. The remaining C (or residue-derived C) within hyphae- or ROOT-ingrowth cores was calculated as the percentage of the initial C content8,21. It is worth noting that any additional C from roots and AMF may affect the final 13C value of the retrieved soil-residue mixture from the field. Because roots and AMF are more 13C-depleted (with δ13C = ca. −27.6‰), the existence of this ROOT/AMF-derived C will make the final mixture even more 13C-depleted. This would theoretically slightly overestimate residue decomposition, that is, underestimate the proportion of residue remaining in soil. However, the impact should be negligible because the quantity of this ROOT-derived C was small (ca. 1.5%) as we determined (ca. 116 g C m−2 soil or 0.58 g C kg−1 soil), compared to total soil C (ca. 37.8 g C kg−1 soil) in the cores (Please see our previous discussion in ref. 21).

Statistical analysis
All analyses were performed using R 4.1.290. The linear mixed model was used to examine the main effects of Nr input, warming and their interaction, and whether these changed over time. To account for repeated measurements, a first-order autoregressive function (corCAR1) in the “nlme” package91 was included in the model. Then, in each year, two-way ANOVAs were conducted to further analyze the effects of Nr input and warming on root, AMF and soil variables, including Nr input and warming as fixed factors and block as a random effect (“lmer” function from the lme4 package)92. Tukey’s multiple comparisons test was performed for comparisons among the four different treatments. Pearson correlations were used to examine the relationship of AMF and root with soil/root variables, and with the dynamics of AMF- and ROOT-derived C. PCA was performed to visualize the root trait and AMF colonization coordination using the “prcomp” function with scaling option in the “stats” package93.

To determine the effects of Nr input and warming on the community composition of AMF in soil and plant roots, PERMANOVA with 9999 permutations was carried out using the “adonis2” function in the package “vegan” in R86. To illustrate the differences in AMF communities in soil and roots between the four treatments, nonmetric multidimensional scaling (NMDS) with Bray–Curtis distances was performed using the “metaMDS” function in the “vegan” package86. The significant differences were determined at the 95% probability level.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The MS titled “Shifts in root traits and mycorrhizal fungal community control reactive N and warming effects on soil organic C” provides an interesting study in the field of soil C dynamics under climate change. Authors by manipulating N input (urea) and warming (open top chambers) concentrate on vegetation change, root traits, microbial communities, and soil C. Their results confirm available data showing that shift in plant root traits and mycorrhiza due to warming and N addition affect soil C.\nI have some doubts about novelty/significance and about methodology and experimental design.\nExperimental design and sampling:\nAuthors established 4 treatments: control, +12 N m-2 per year, ambient temp. and warming. Why only one +N treatment was established? Experimental design would benefit from 2-3 N levels. (line 302)\nSampling: soil 0-10cm was sampled – why it was not separated into organic vs mineral soil? (line 325).\nSoil storing (line 332) – soil was stored in +4C for two weeks. Why so long? Significant changes in sieved soil could take place after two weeks.\nMethodological aspects:\nsoil inorganic N (line 336) soil was extracted with K2SO4 for inorganic N analyses. Why not with 1M KCl which is commonly used for extraction of inorganic N?\nAMF (arbuscular mycorrhiza) biomass in the soil (line 371): Authors used for AMF biomass in the soil PLFA analyses in line with protocol from 1993. However, newer studies proved that PLFA (and its AMF biomarker) is not good representative of AMF, and NLFA should be used instead (see for example 10.1155/2012/236807)\nMAOM: (line 482) – mineral associated organic matter (called in figures MAOC) was used to determine C stability. It is of rather limited value. As mentioned in introduction (line 72) microbial necromass is important for C stabilization, why it was not measured? Moreover, MS would benefit from more in-depth studies of C fractions, e.g. labile vs stabile C pools, glomalin (as it is produced by AMF and it is assumed as stable).\nMoreover, MAOM does not necessary represent stable C pool, as root exudates may easily access mineral-associated matter (see for example work by Keiluweit et al. 2015, 10.1038/NCLIMATE2580), thus using MAOM as only marker of soil C stability is maybe not the best choice.\nSignificance:\nOn the basis of abstract, I was assuming that the study was done not only at one site with limited amount of treatments. Some results may be site-specific or emerge from limited amount of treatments (N addition at only one level). I cannot see anything specific for this MS comparing to existing literature. Thus, main novelty of this MS should be better marked in the text and in the abstract.", "reviewer_2": "The authors present a well-designed, thorough, multi-year study that investigates the effects of nitrogen enrichment and warming on root traits, mycorrhizal dynamics, and soil carbon cycling. The findings are interesting and would make a valuable addition to the literature. However, after reading the manuscript a few times, I am still struggling to identify the main findings that will inform future research. This is not due to a lack of interesting findings, but rather to the impressive array of experimental components/results and complexity of Nr/warming effects on root/mycorrhizal/C dynamics, which make it difficult to synthesize the results into a simple message. I think that the manuscript will be more impactful if the authors are able to pare down or reframe the results in a way that better highlights the main findings. It may be helpful to separate the results/discussion into smaller sections with subheadings that summarize each main finding. A conceptual diagram may also be helpful.\n1-2 and 39-43: Perhaps I am misunderstanding these sentences, but to me, the language used here implies a causal/definitive mechanism that dominates over others and persists over long timescales. I think that this overstates your findings a bit. Please revise.\n58-59: I think that something is missing from this sentence.\n145-148: Based on my understanding, these changes are due to a shift in plant community composition rather than a shift within each plant species. If that is correct, please modify the sentence so that this is clear. Also, what is your interpretation of the effects of warming?\n191-193: Do you account for non-mycorrhizal fungi that may also transport C from the root or rhizosphere into the 25-micron mesh ingrowth cores? Please address this somewhere within the manuscript.\n227-228: This statement contains several assumptions. Please support these assumptions with your own data or with published literature.\n254-257: Rather than speculate about this, why not interpret this within the context of your results? Were Nr input and fine root turnover rates positively associated with decomposition?\n361-363: It would be helpful to provide an explicit definition for each abbreviation and acronym (eg, V, A, L).\n363-364: Where the dried roots used for the assay described in lines 365-370? If so, were the roots rehydrated before clearing/staining?\n365-370: Were the roots fixed before clearing? At what temperature were the roots cleared, acidified, and stained? Please provide the concentration of the glycerol/trypan blue solution. Please indicate the total root length and number of intersections assessed per sample.\n444-452: Was -27.45 per mil used as the delta 13C value for all root samples? If so, why not use the value from each respective core? Is this calculation conducted separately for the AMF and root ingrowth cores? Please specify (as in Zhang et al 2019). It is not clear from the notation alone (“delta 13C root/mycelium”). Is there evidence that the -1.5 per mil correction is valid for AMF? Do you account for non-mycorrhizal fungi that may also transport C from the root or rhizosphere?\n472-474: Please provide any details/context that the reader will need in order to understand your experiment and results.\n479-480: At what temperature were the soils dried?\n495: What was the concentration of the DCB?\n496: At least 16h? Were some soils shaken for longer? If so, which soils and how much longer?\n497-499: I don’t entirely understand this sentence or the motivation for this procedure. Is this to account for the influence of C in DCB or sodium dithionite? What was the concentration of the sodium dithionite?\nFigures: For all figures in main manuscript and SI:\n- Please choose a color-blind friendly palette or use symbols, linetypes, or fill texture to differentiate between treatments.\n- Please add definitions of the relevant significance levels to all figures. For example, *** is missing from Fig 1, the cross is missing from Fig 2, and none of the symbols are defined in Fig S7.\n- Please define all elements of each figure. For example, in Fig S9—please indicate what the linetype represents (perhaps this can replace the sentence on 105-106 that describes significance symbols that are not used in this figure; same for Fig S10). Fig S11—please specific what the colors represent.\n- In many of the bargraphs it is difficult to interpret the effect of the treatments. Can you add a compact letter display to indicate statistically significant differences between treatments? I think that this would make it much easier for readers to identify differences.\nTables: Please indicate what the italicization of numbers represents.\nFig S3: Please add the years to the figure (as in Fig S5). Please add error bars to each point and indicate if any differences were statistically significant in the figure caption.", "reviewer_3": "General comments: The manuscript provides substantial evidence for warming and N addition control on the role that fine roots and arbuscular mycorrhizal fungal (AMF) traits play in forming soil carbon (C). Overall, this is a well-written paper that is easy to read. However, there are a few significant concerns. First, the novelty of the study design needs to be better articulated in the introduction. There have been many studies that have evaluated the effects of warming or N addition alone on the parameters measured in this study. This study is unique in that it includes a combination treatment (warming x N addition); however, the authors do not highlight this at all in the introduction. The simultaneous application of warming and nitrogen (N) addition has been shown to elicit synergistic effects when in combination that are otherwise not observed in single-factor treatments (warming and N alone). The authors thus miss a critical opportunity to highlight the significance of their study.\nSecond, there is a potential major flaw with the mineral soil C data. It appears that all soil was dried at 65°C. However, standard methods for determining gravimetric water content use 105°C for mineral soils (65°C is used for plant materials and organic soil horizons). Thus, there is likely an underestimate of soil water content and an overestimate of soil C. This is especially problematic in a study like this where there are differential treatment differences in soil moisture (i.e., warming will dry the soil relative to the control and N addition plots).\nThere is also an over-interpretation of the fine root and AMF trait data. The authors suggest that changes in plant adaptive responses to global change influence soil C. It remains unclear whether the plant response was due to plasticity at the individual level or a plant community composition shift (which the authors report as occurring). In all cases, this is likely not an adaptive response and is more likely a combination of acclimation at the species level and a shift in community composition. Regarding the ingrowth core experiment, it needs to be clarified how root-derived C was calculated. For example, because roots cannot be isolated from AMF, AMF-C should be subtracted from the total C deposited in the core to get a true measure of root-derived C. Was this done? Additionally, there were no dissolved organic C (DOC) control cores reported. A significant portion of new C deposited into the core could be from DOC inputs via rain and soil water lateral flow. In my experience, warmed soils show significantly more DOC contamination using this method.\nFinally, no interaction term (warming x N addition) is reported. You need to test for this interaction to appropriately determine the significance of your treatments given the field design (full factorial). Also, it doesn’t appear that the statistical approach used accounted for the fact that measurements were made over time in the same plots (i.e., repeated measures)? See below for more detailed comments about each of these (and other) issues.\nSpecific line comments:\nL59. Many studies have found that N addition suppresses decomposition, contributing to the accumulation of soil C. Changes to the N cycle need to be seriously considered in this study, rather than assuming that N increases decomposition or is not a driving factor in warmed soils. Additionally, not all plants respond positively to N addition, and in some cases, plant growth itself is suppressed. A more thorough review and citation of the literature on N addition impacts on plant growth and decomposition is warranted. If considered through the lens of suppressed decomposition, the interpretations of this study would change entirely.\nL62. A major theme of the paper is the effects of N and warming on plant dynamics. Nitrogen availability often co-varies with soil warming. That is, warming increases N mineralization rates and thus N availability. This study could directly parse out the effects of warming versus N addition. For example, you can comment on whether warming effects on the ecosystem are due to changes in water or N dynamics. The novelty of the study has yet to be sufficiently conveyed.\nL67. This is relevant to mycorrhizae generally and not just AMF. Since the study system has not yet been defined, it’s important to be more general or to describe why you are focusing on AMF specifically.\nL76. The term “priming” has a specific definition which is that labile plant C inputs stimulate the decomposition of native soil organic matter. Clarify the language here.\nL80. This assumes that N addition equally affects the members of a community, which may not be a valid assumption. Does adding N favor certain grasses or forbs based on different root foraging strategies?\nL92. Water is not the only factor that warming influences. As noted above, there should be careful consideration of changes in N cycling under warming. Can you provide information on N availability in response to the treatments? Was there any evidence of a drought by plants? Otherwise, it is difficult to attribute the warming treatment to water deficit without having a warming x drought treatment. But you can comment on whether warming and N addition plots had similar effects. This will allow you to at least be more confident that the effects were due to water and not N.\nL104-106. The first hypothesis is not a strong scientific hypothesis in that it isn’t directional (i.e., how specifically are N inputs predicted to shift the plant community?) and no mechanisms is provided. Having said that, I’m not in favor of writing hypotheses after the data are in hand, since that then influences how the hypothesis is written.\nL106. The phrase “priming residue decomposition” is imprecise and unclear. Although I understand what you mean, someone unfamiliar with the priming process may not.\nL130. Can you tell whether this was due to the change in plant community? For those unfamiliar with this ecosystem, do the new species produce more biomass or have invasive-like qualities?\nL139. Root turnover in your study was primarily a function of root biomass, not production. i.e., turnover was driven by standing biomass, and I am skeptical of how accurately this reflects true turnover.\nL200. I understand why this is called AMF-derived C, but it may also include other fungi.\nL225. Clarify whether these were traits measured from bulk soil or soil from the ingrowth core.\nL251. Terms like fragile are vague. Is there a more quantitative way to describe roots that turnover faster?\nL255. You can’t be certain that this would increase root exudate inputs. I would instead change language in the text to generally say “may increase root-derived C”\nL256. Counterintuitive. Earlier, the paper stated that the plants and microbes were N and P limited. This needs to be clarified.\nL366. How many root tips were counted? Often hundreds of tips need to be counted to determine the colonization rate successfully. Clarify.\nL423. How were DOC inputs determined?\nL452 Clarify if AMF-C was subtracted from root C recovered in cores. If not, you should change the language to “root + fungal-derived C“, since both roots and hyphae grew into the cores.\nL461. Is this a standard method?\nL483. It is standard protocol to dry mineral soils at 105°C to remove soil pore water, thus soil moisture content is very likely underestimated which then influences soil C content (likely an overestimate). Since warming influences soil moisture, this could influence the relative differences between treatments.\nL502. Was the interaction between warming and N addition tested? It is not reported in the methods, nor does this show in any figure.\nL502. I’m concerned about the importance of time in this study since you make repeated measures in the same plots. Your model did not include time, which could be a significant factor in structuring the root traits and soil C patterns. For example, did each year have similar weather patterns? Was the heating consistent each year? Other environmental variables may vary over time, and thus time would be an important factor to account for this variability. It would also be good to know in which year the biggest changes occurred. If time is insignificant in your model, you may proceed with ANOVA as you have conducted them, but the effect of time should be noted.\nL739. There is a lot of repetition in the figure. One suggestion would be to keep the matrix and the PCA and move the individual variables that comprise the PCA to a supplemental table.\nL758. Is there a significant interaction between warming and N addition?"}, "author_response": "Point by point responses to Reviewers’ Comments on Manuscript\nNature Communications (NCOMMS-23-28603A-Z)\n(Comments in black and Responses in blue)\nReviewer #1 (Remarks to the Author):\nThe MS titled “Shifts in root traits and mycorrhizal fungal community control reactive\nN and warming effects on soil organic C” provides an interesting study in the field of\nsoil C dynamics under climate change. Authors by manipulating N input (urea) and\nwarming (open top chambers) concentrate on vegetation change, root traits, microbial\ncommunities, and soil C. Their results confirm available data showing that shift in plant\nroot traits and mycorrhiza due to warming and N addition affect soil C.\nI have some doubts about novelty/significance and about methodology and\nexperimental design.\nReply1-1: We appreciate your interest and constructive comments on our manuscript.\nWe agree that the previous manuscript did not show sufficient novelty. However, we\nfeel that the major reason is that we did not develop the manuscript well to clearly\nillustrate the novel aspects of our work, rather than the study lacks novelty.\nTo our knowledge, our study represents the first comprehensive examination of the\nlinkages between root traits/arbuscular mycorrhizal fungal (AMF) community and soil\nC dynamics as influenced by two pervasive climate change drivers, reactive N input\nand warming, in a natural ecosystem setting. Over the last several decades, increasing\nexperimental evidence has shown that N input and warming alter plant C allocation to\nroots and their symbiotic mycorrhizal fungi, modifying root traits and AMF community\ncomposition (see meta-analyses, e.g., Li et al. 2015; Han et al. 2020; Wang et al. 2021;\nHu et al. 2022). However, the resulting impact on soil C dynamics and the underlying\nmechanisms remain largely unknown.\nBy integrating long-term field manipulation, 13C stable isotope tracing and MiSeq\nsequencing, our work established the mechanistic linkages among N input and warming,\nroot traits/AMF community composition, and organic C decomposition and stability,\nwhich we believe is highly novel. We have now modified our manuscript accordingly\nto illustrate the novelty of our work. Please find a point-by-point response to your\ncomments and suggestions below.\nExperimental design and sampling:\nAuthors established 4 treatments: control, +12 N m-2 per year, ambient temp. and\nwarming. Why only one +N treatment was established? Experimental design would\nbenefit from 2-3 N levels. (line 302)\nReply1-2: Insightful comment. We would agree that experimental design would benefit\nfrom 2-3 N inputs. In this study, we focused on one high N input level (12 g N m-2 yr-\n1) based on the following reasons.\nFirst, we applied urea to simulate fertilization because applications of N fertilizers are\nroutine in many areas of Loess Plateau to increase yield of forage grasses and crop\nplants. Our N inputs were high compared to the natural N deposition, but were within\nthe range of which N fertilizers are applied to enhance grass productivity (Wei et al.\n2018; Chen et al. 2021). For example, in a long-term fenced grassland on the Loess\nPlateau, Wei et al. (2018) reported that high N inputs (20.0 g N m−2 yr−1), but not low\nN addition (10.0 g N m−2 yr−1), significantly enhanced aboveground plant biomass.\nSecond, we have an ongoing N addition gradient experiment adjacent to our field site.\nThis experiment has 5 N input levels (0, 1, 4, 8, and 12 g N m-2 yr-1) and we found that\nlow to moderate N inputs (1, 4 and 8 g N m-2 yr-1) did not have significant effects on\nplant growth (He, Qiu, Hu et al. unpublished; see Fig. R1 below), but high N inputs (12\ng N m−2 yr−1) significantly altered plant community composition favoring the growth\nof grasses over forbs.\nGiven these considerations, in this study, we chose the N input rate of 12 g N m-2 yr-1.\nResponse Figure 1: Effects of Nr inputs on plant biomass (He et al. unpublished).\na Grass relative biomass. b Forb relative biomass. c Grass to forb biomass ratio. Data\nshow mean ± SE (n = 4). Significant differences as determined by one-way ANOVA\n(*p < 0.05, **p < 0.01) and two-sided Tukey’s HSD are indicated by different letters.\nColoursrepresent different N input treatments.\nSampling: soil 0-10cm was sampled – why it was not separated into organic vs mineral\nsoil? (line 325).\nReply1-3: There was not an organic layer at our experimental site. Prior to the year\n1986, all the plants had been harvested by farmers as fuels or animal forage for nearly\n30 years.\nAlso, it needs to note that in this study, to mimic grazing in the grassland, we clipped\naboveground plant parts once a year at the end of the plant growing period in late\nAugust, and the clippings were manually removed from each plot. Therefore, in our\nfield site, plant residues were not accumulated in the soil to form an organic soil layer.\nWe have now added the information in our Method (see Line 364-366 and 392-394).\nSoil storing (line 332) – soil was stored in +4C for two weeks. Why so long? Significant\nchanges in sieved soil could take place after two weeks.\nReply1-4: Thank you for raising this concern. We would like to emphasize that “The\nsoil samples were kept at 4 ºC, and all the chemical and microbial analyses were\nconducted or initialized within two weeks”. It is not that we stored soils for two weeks\nbefore we started analyzing them. Analyses for some microbial parameters require\nmultiple weeks, such as soil respiration (2-wk incubation) and N mineralization (4-wk\nincubation). That is why we used the word “initialized”.\nAlso, we would like to mention that our experimental site is in remote Northwest China\nwhich is ca. 1500 km far from our lab at Nanjing. It often takes 2-4 days to ship our\nsamples to the lab for processing. Second, soil samples need to be sieved to remove\nrocks and roots and get representative subsamples for microbial analyses. We always\nstarted analyzing microbial parameters right after the soil was sieved, considering that\nmicrobial parameters can change quickly, as you rightfully pointed out (see Line 395-\n398).\nMethodological aspects:\nsoil inorganic N (line 336) soil was extracted with K2SO4 for inorganic N analyses.\nWhy not with 1M KCl which is commonly used for extraction of inorganic N?\nReply1-5: Good point. It is true that for extraction of mineral N, 1M KCl is used more\nfrequently than 0.5M K SO . Yet, the principle is the same: K+ replacing NH + and Cl-\n2 4 4\n(or SO 2-) replacing NO -. In either case, K+ and Cl- (or SO 2-) are in excess to ensure\n4 3 4\nthat they completely replace NH + or NO - from soil colloids (by the way, very little\n4 3\nanion adsorption occurs on soil colloids anyway).\nAMF (arbuscular mycorrhiza) biomass in the soil (line 371): Authors used for AMF\nbiomass in the soil PLFA analyses in line with protocol from 1993. However, newer\nstudies proved that PLFA (and its AMF biomarker) is not good representative of AMF,\nand NLFA should be used instead (see for example 10.1155/2012/236807)\nReply1-6: This is a very good point. Because AMF are obligately biotrophic,\nquantifying AMF (biomass and community composition) and their impact on soil C in\nfield is particularly challenging. Indeed, none of the existing methods is perfect or can\ndescribe the whole picture of AMF in soil. That is why we used three different methods\nto characterize AMF, namely, microscopic determination of AMF colonization in roots,\nPLFA for external AMF biomass in soil, and MiSeq sequencing for AMF community\ncomposition and relative abundance of different AMF families. We are aware that it has\nbeen reported that PLFA may not be the best method for soil AMF biomass (Ngosong,\nGabriel and Ruess, 2012.), but the PLFA method is still popularly used. We also noticed\nthat both authors for this paper, Dr. Ngosong and Dr. Ruess, have used the PLFA method\nin recent years (e.g., Faust et al. 2017; Rodríguez et al. 2022). Indeed, Faust S, Heinze\nS, Ngosong C et al. (2017) argued that “This indicates negligible interference from\nbacteria as the ratio of NLFA/PLFA 16:1ω5 is larger than 1, suggesting that PLFA\n16:1ω5 is a suitable marker for AMF biomass in soil”.\nAlso, we have now measured NLFA of our soil samples and we found that the ratio of\nNLFA/PLFA 16:1ω5 is around 2 (see Figure R2a below), suggesting that the\ncontribution of bacterial PLFA was low and the PLFA method produce reasonably\nestimates of AMF biomass in our study. Our results showed that N input and warming\nsignificantly reduced AMF NLFA in 2021 (see Figure R2b below). Last but not least,\nwe did not ONLY focus on the AMF biomass based on PLFA in our manuscript. Rather,\nwe focused on the AMF community composition and relative abundances of dominant\nfamilies. Together, we feel that our approach measuring multiple parameters provides\na more comprehensive picture of AMF in soil and roots.\nResponse Figure 2: Effects of Nr input and warming on AMF NLFA/PLFA ratio\nand NLFA biomass in 2021. a AMF NLFA/PLFA ratio. b AMF NLFA. Data show\nmean ± SE (n = 4). Significant differences as determined by two-way ANOVA (†p <\n0.1, *p < 0.05, **p < 0.01 and ***p < 0.001) and two-sided Tukey’s HSD are indicated\nby different letters. Coloursrepresent different treatments. Con represents control; N\nrepresents N input; W represents warming only; and N+W represents combined N input\nand warming.\nMAOM: (line 482) – mineral associated organic matter (called in figures MAOC) was\nused to determine C stability. It is of rather limited value. As mentioned in introduction\n(line 72) microbial necromass is important for C stabilization, why it was not measured?\nReply1-7: Good point. We have now measured microbial necromass (Line 538-552)\nand added the result in our Main Text (see Figure R3 below or Fig. 5c). Result showed\nthat compared with the control, Nr input, warming and their interaction significantly\nreduced microbial necromass C by 11.4, 10.5 and 15.0%, respectively. However, the\npresence of AMF alone and roots with AMF had no effect on microbial necromass\n(Line 257-260).\nResponse Figure 3: Nr input and warming modulated the effects of AMF and plant\nroots on microbial necromass C after 2-yr incubation. Data show mean ± SE (n = 4).\nSignificant differences as determined by three-way ANOVA (**p < 0.01 and ***p <\n0.001) and two-sided Tukey’s HSD are indicated by different letters. Uppercase letters\nabove bars indicate significant differences among four climate change treatments. Con\nrepresents control; N represents N input; W represents warming only; and N+W\nrepresents combined N input and warming. ROOT refers to roots with AMF.\nMoreover, MS would benefit from more in-depth studies of C fractions, e.g. labile vs\nstabile C pools, glomalin (as it is produced by AMF and it is assumed as stable).\nReply1-8: We have measured soil labile C (dissolved organic C, DOC; Line 401-405)\nand added the result in our Supplementary Information (see Figure R4 below or Fig.\nS12a).\nHowever, we did not measure soil glomalin because the nature of glomalin itself is still\nvery controversial (Irving et al. 2021): it is generally believed that glomalin includes\nmultiple secondary compounds or metabolic products of AMF, but not a structure\ncomponent of fungal cells, which means that converting glomalin into AMF biomass is\nvery challenging, if not impossible. Equally important, glomalin can stay in soil for a\nlong time, and we do not know whether the glomalin was residues from previous years\nor products from the alive AMF in the current growing season.\nResponse Figure 4: Nr input and warming modulated the effects of AMF and plant\nroots on DOC after 2-yr incubation. Data show mean ± SE (n = 4). Significant\ndifferences as determined by three-way ANOVA (**p < 0.01). Uppercase letters above\nbars indicate significant differences among four climate change treatments, and\nlowercase letters indicate, for each climate change treatment, significant differences\namong mycorrhizae treatments (one-way ANOVA, Tukey’s HSD, two-sided). Con\nrepresents control; N represents N input; W represents warming only; and N+W\nrepresents combined N input and warming. ROOT refers to roots with AMF.\nMoreover, MAOM does not necessary represent stable C pool, as root exudates may\neasily access mineral-associated matter (see for example work by Keiluweit et al. 2015,\n10.1038/NCLIMATE2580), thus using MAOM as only marker of soil C stability is\nmaybe not the best choice.\nReply1-9: This comment is legitimate. In principle, the stable C pool we often called\nis not exactly stable because “the stable C” is in dynamics. Mineral associated organic\nmatter is largely made of microbial products richer in N and persists in soil because of\nchemical bonding to minerals and physical protection in small aggregates (Cotrufo et\nal. 2019). Yes, some mineral-associated organic matter (MAOM) can be displaced by\nroot exudates (Keiluweit et al. 2015) and turn over on short timescales (Jilling et al.\n2018), particularly when high amounts of root exudates or other labile C are added. Yet,\nthe majority of MAOM persists for hundreds or thousands of years before turning over\n(Lützow et al. 2006; Cotrufo et al. 2019), forming a functionally important pool for\nlong term SOC sequestration.\nTo address this concern more thoroughly, we have now included microbial necromass\nC as an additional indicator of persistent soil organic matter (see Reply 1-7 for details),\nwhich complements MAOM and allows a more robust assessment of SOC stability in\nresponse to Nr input and warming.\nSignificance:\nOn the basis of abstract, I was assuming that the study was done not only at one site\nwith limited amount of treatments. Some results may be site-specific or emerge from\nlimited amount of treatments (N addition at only one level). I cannot see anything\nspecific for this MS comparing to existing literature. Thus, main novelty of this MS\nshould be better marked in the text and in the abstract.\nReply1-10: Thank you for this insightful comment. Sorry that we should have added\nmore background on the novelty of our study.\nWhile it is true that the experiment was conducted at a single site with one N addition\nlevel, our study goes beyond standard field manipulations by integrating multiple\nadvanced methodologies (e.g., long-term factorial field treatments, 13C stable\nisotope tracing, PLFA/NLFA analysis, microscopic root colonization, and high-\nthroughput MiSeq sequencing) to explore the mechanistic linkages among warming\nand N input, plant communities, root traits/AMF, and soil C decomposition and\nstabilization.\nAs increasing experimental evidence has shown that warming and N input alter plant C\nallocation to roots and their symbiotic mycorrhizal fungi, modifying root traits and\nAMF community composition (see meta-analyses, e.g., Li et al. 2015; Han et al. 2020;\nWang et al. 2021; Hu et al. 2022). However, the resulting impact on soil C dynamics\nand the underlying mechanisms remain largely unknown. Our study is, to our\nknowledge, the first to explicitly link trait-based plant-microbe responses to two\npervasive global change drivers (Nr input and warming) with stable and labile soil C\npools, including microbial necromass C, which we believe is highly novel.\nWe have now substantially revised the abstract and Main text to better present the\nnovelty of our study.\nReviewer #2 (Remarks to the Author):\nThe authors present a well-designed, thorough, multi-year study that investigates the\neffects of nitrogen enrichment and warming on root traits, mycorrhizal dynamics, and\nsoil carbon cycling. The findings are interesting and would make a valuable addition to\nthe literature. However, after reading the manuscript a few times, I am still struggling\nto identify the main findings that will inform future research. This is not due to a lack\nof interesting findings, but rather to the impressive array of experimental\ncomponents/results and complexity of Nr/warming effects on root/mycorrhizal/C\ndynamics, which make it difficult to synthesize the results into a simple message. I\nthink that the manuscript will be more impactful if the authors are able to pare down or\nreframe the results in a way that better highlights the main findings. It may be helpful\nto separate the results/discussion into smaller sections with subheadings that summarize\neach main finding. A conceptual diagram may also be helpful.\nReply2-1: Thanks for your supports and constructive comments. We have now\nreframed our results and separated Discussion into small sections with subheadings to\nbetter highlight and summarize our main findings. Also, we modified our conceptual\ndiagram to better convey our key findings (Fig. 6). Please see our detailed\nresponses/explanations below.\n1-2 and 39-43: Perhaps I am misunderstanding these sentences, but to me, the language\nused here implies a causal/definitive mechanism that dominates over others and persists\nover long timescales. I think that this overstates your findings a bit. Please revise.\nReply2-2: Great comment. We have now revised the sentences (see Line 1-2 and 53-\n56).\n“Root traits and mycorrhizal fungal community mediate reactive N and warming\nimpacts on soil organic carbon.”\n“Together, these findings provide the mechanistic evidence linking two pervasive\nglobal change drivers (Nr input and warming) to root-AMF interactions and soil C\ndynamics in a natural ecosystem, highlighting plant root traits and AMF composition\nas predictive indicators for long-term soil C balance under future climate change\nscenarios.”\n58-59: I think that something is missing from this sentence.\nReply2-3: Thanks for pointing this out. We have now modified this sentence (Line 76-\n78).\n145-148: Based on my understanding, these changes are due to a shift in plant\ncommunity composition rather than a shift within each plant species. If that is correct,\nplease modify the sentence so that this is clear. Also, what is your interpretation of the\neffects of warming?\nReply2-4: Insightful comment. We have now modified the sentence to better interpret\nour results (see Line 191-194).\n“These tradeoffs between root length/surface area and root density/diameter reflect\nshifts in plant C allocation strategy (via altering plant community composition) that\nfavor long, low-density roots with low C investment under Nr input while construct\nthicker but shorter roots under warming.”\n191-193: Do you account for non-mycorrhizal fungi that may also transport C from the\nroot or rhizosphere into the 25-micron mesh ingrowth cores? Please address this\nsomewhere within the manuscript.\nReply2-5: Insightful comment. No, we did not account for C transported from non-\nmycorrhizal fungi. The 25-micron mesh ingrowth cores may allow non-mycorrhizal\nfungi to transport C into the cores. Yet, because saprotrophic fungi usually grow on and\ndecompose the residues, but do not transport C from low-C soil to residues, their\ntransportation of C into the ingrowth cores should be limited.\nFurther, in May 2018, we installed δ13C-enriched ingrowth cores filled with C plant\n4\nresidue (switchgrass) and soil/sand (1:5/w:w) mixture (with a final δ13C = -15.52‰) to\nreduce soil C in ingrowth cores and minimize the growth of saprotrophic fungi\nfollowing the method in Wallander et al. (2001) and Jach-Smith and Jackson (2017).\nThis approach helped reduce background soil C and discouraged saprotroph\nproliferation.\nMoreover, our results showed that N input and warming significantly reduced AMF-\nand ROOT (or root+AMF)-derived C accumulation (see Figure. R5 below). Therefore,\nwe mainly focused on AMF-derived C in this study. We have now added the\ninformation in our Method.\nResponse Figure 5: N input and warming modulated the effects of AMF- and/or\nROOT-derived C accumulation to soil in 2018. Data show mean ± SE (n = 4).\nSignificant differences as determined by two-way ANOVA (**p < 0.01 and ***p <\n0.001) and two-sided Tukey’s HSD are indicated by different letters. Con represents\ncontrol; N represents N input; W represents warming only; and N+W represents\ncombined N input and warming. ROOT refers to roots with AMF.\n227-228: This statement contains several assumptions. Please support these\nassumptions with your own data or with published literature.\nReply2-6: Good point. We have now added references of Davidson & Janssens (2006)\nand Li Z et al. (2021) to support our assumptions that warming-induced water stress on\nsoil microbes offset the priming effect of increased root C on microbes (Line 277).\n254-257: Rather than speculate about this, why not interpret this within the context of\nyour results? Were Nr input and fine root turnover rates positively associated with\ndecomposition?\nReply2-7: Good comment. We have now performed linear regression analysis to\ndetermine the relationship between fine root turnover and decomposition. Our results\nshowed that fine root turnover was significantly negatively correlated with residue-\nderived C remaining in the ROOT-ingrowth cores (see Figure R6 below), that is, root\nturnover positively correlated with the decomposition rate in the root-ingrowth core.\nWe have now added the results in the Supplementary Information (Fig. S19e) and\nmodified the sentence (Line 320-324).\nResponse Figure 6: Nr input and warming modulated the relationships between root\nturnover and residue-derived C remaining in ROOT-ingrowth cores. ROOT refers to\nroots with AMF.\n361-363: It would be helpful to provide an explicit definition for each abbreviation and\nacronym (eg, V, A, L).\nReply2-8: Sorry for the confusion. We have now added an explicit definition for each\nabbreviation in the Method (see Line 422-425).\n363-364: Where the dried roots used for the assay described in lines 365-370? If so,\nwere the roots rehydrated before clearing/staining?\nReply2-9: Yes, dried roots were rehydrated in deionized water for ca. 10 hours before\nclearing. We have now added the information (Line 427-429).\n365-370: Were the roots fixed before clearing? At what temperature were the roots\ncleared, acidified, and stained? Please provide the concentration of the glycerol/trypan\nblue solution. Please indicate the total root length and number of intersections assessed\nper sample.\nReply2-10: Thank you for your detailed questions. Our root samples were not fixed\nbefore clearing. For the AMF infection determination, “dried roots from each plot were\ncut into ~1 cm segments, and approximately 100 segments were randomly chosen and\nrehydrated in deionized water for ca. 10 hours. The rehydrated root samples were then\ncleared in 10% (w/v) KOH at 90 °C for 30 min, and acidified in 1% (v/v) HCl for 1 h.\nThe root segments were subsequently stained in an acidic glycerol-trypan blue solution\n(50% glycerol (v/v), 1% hydrochloric acid (v/v), and 0.05% trypan blue (w/v)) and\nheated for 30 min in a steamer. Stained roots were fully washed with water and spread\non a Petri dish with gridlines, and examined for AMF colonization under ×40\nmagnification by counting ca. 100 intersects per sample, using a stereomicroscope\n(SZN71, Sunny Optical Technology Co., Ltd, China)” (see Line 427-435).\nWe have now added the concentration details of the staining solution and clarified the\nprocedures in the Methods section. Information regarding the total root length and the\nnumber of intersections assessed per sample has also been added for completeness (see\nLine 427-435).\n444-452: Was -27.45 per mil used as the delta 13C value for all root samples? If so,\nwhy not use the value from each respective core?\nReply2-11: Yes, a uniform δ¹³C value of -27.45 per mil was previously used for all root\nsamples. To address this concern, we have now measured the δ¹³C value for root\nsamples of each treatment. As expected, there were no significant effects of Nr input\nand warming on root δ13C (see Figure R7).\nAlso, we have now used the δ13C value of each root sample grown into each respective\ncore to calculate ROOT- and/or AMF-derived C. The updated results showed almost\nsame with our previous manuscript, confirming the robustness of our findings.\nResponse Figure 7: Effects of Nr input and warming on root δ13C in 2021. Data show\nmean ± SE (n = 4). No significant difference was observed among different treatments.\nCon represents control; N represents N input; W represents warming only; and N+W\nrepresents combined N input and warming. ROOT refers to roots with AMF.\nIs this calculation conducted separately for the AMF and root ingrowth cores? Please\nspecify (as in Zhang et al 2019). It is not clear from the notation alone (“delta 13C\nroot/mycelium”). Is there evidence that the -1.5 per mil correction is valid for AMF?\nReply2-12: Thank you for raising this important point. We apologize for the confusion.\nThe publication of Zhang et al. (2019) we previously cited was inappropriate, as that\nstudy focused on ectomycorrhizal fungi rather than arbuscular mycorrhizal fungi\n(AMF). They obtained δ13C of ectomycorrhizal fungi by performing a\nmycelium\nconservative isotopic correction (+1.5‰) from δ13C , which was not directly\nroot\napplicable to AMF.\nBecause it is quite difficult and challenging (if not possible) to extract AMF hyphae in\nfield and then determine the δ13C value (Walder et al. 2013), we have now assumed that\nthe signature of AMF equals that of roots, as it was often used in other studies (see\nWalder et al. 2013; Huang et al. 2020; Zhang et al. 2020) (see Line 500-501). Indeed,\nZhang et al. (2020) stated that “We assumed that the 13C of the AMF equals the 13C of\nthe fine roots”.\nWe have now removed the reference of Zhang et al. (2019) and clarified this assumption\nin the manuscript. Importantly, using root-specific δ13C values instead of a corrected\nAMF estimate did not alter our key findings or conclusions.\nDo you account for non-mycorrhizal fungi that may also transport C from the root or\nrhizosphere?\nReply2-13: We did not account for non-mycorrhizal fungi that may also transport C\nfrom the root or rhizosphere because of the reasons explained in Reply 2-5.\n472-474: Please provide any details/context that the reader will need in order to\nunderstand your experiment and results.\nReply2-14: Good suggestion. We have now added more details into the Main Text to\nensure the reader to understand the experiment and results. The following details have\nbeen incorporated:\n“The plant community at our field site has been long dominated by C plants and the\n3\nsoil exhibits a depleted 13C signature with δ13C = -23.5‰ (see Line 240-241).”\n“To assess the influence of roots and AMF on residue decomposition in the\nmycorrhizosphere, ingrowth cores were prepared in September 2018. Briefly, we placed\n1.80 g of ca. 1 cm length dried maize litter (δ13C = -11.58‰) into each core through\nmixing with 150.0 g of field soil (δ13C = -23.49‰) following the method in our previous\npublications (Cheng et al. 2012; Qiu et al. 2021)” (see Line 554-557).\nAdditionally, we have provided details on how mycorrhizal and root C may affect the\nfinal 13C value of the soil-residue mixture.\n“Because roots and AMF are more 13C-depleted (with δ13C = ca. -27.6‰), the existence\nof this ROOT/AMF-derived C will make final mixture even more 13C-depleted. This\nwould theoretically slightly overestimate residue decomposition, that is, underestimate\nthe proportion of residue remaining in soil. However, the impact should be negligible\nbecause the quantity of this ROOT-derived C was small (ca. 1.5%) as we determined\n(ca. 116 g C m-2 soil or 0.58 g C kg-1 soil), compared to total soil C (ca. 37.8 g C kg-1\nsoil) in the cores (Please see our previous discussion in Qiu et al. 2021) (see Line 567-\n573).”\n479-480: At what temperature were the soils dried?\nReply2-15: We oven-dried soil samples to a constant weight at 65 °C for 72 h (Line\n518).\n495: What was the concentration of the DCB?\nReply2-16: DCB containing 0.27 M trisodium citrate, 0.11 M sodium bicarbonate and\n0.1 M sodium dithionite was used to extract Fe-associated organic C.\n496: At least 16h? Were some soils shaken for longer? If so, which soils and how much\nlonger?\nReply2-17: Sorry that it was a typo: it should be 16 hours, and the sentence is that “The\nsuspension was vortexed for 1 min, shaken for 16 h, and centrifugated at 3000 rmp for\n10 min” (Line 534-535).\n497-499: I don’t entirely understand this sentence or the motivation for this procedure.\nIs this to account for the influence of C in DCB or sodium dithionite? What was the\nconcentration of the sodium dithionite?\nReply2-18: Sorry for the mistake. It should be that “Several blanks only containing\nDCB (0.27 M trisodium citrate, 0.11 M sodium bicarbonate and 0.1 M sodium\ndithionite) and deionized water were determined to correct C concentrations in the\nsamples to eliminate the influence of C in DCB” (Line 532-537).\nFigures: For all figures in main manuscript and SI:\n- Please choose a color-blind friendly palette or use symbols, linetypes, or fill texture\nto differentiate between treatments.\nReply2-19: Thanks. We have now modified all the figures to better differentiate\ntreatments using color scheme that is color-blind safe.\n- Please add definitions of the relevant significance levels to all figures. For example,\n*** is missing from Fig 1, the cross is missing from Fig 2, and none of the symbols are\ndefined in Fig S7.\nReply2-20: Thanks for pointing this out. We have now carefully checked each figure\nand added definitions of the relevant significance levels to each figure.\n- Please define all elements of each figure. For example, in Fig S9—please indicate\nwhat the linetype represents (perhaps this can replace the sentence on 105-106 that\ndescribes significance symbols that are not used in this figure; same for Fig S10). Fig\nS11—please specific what the colors represent.\nReply2-21: Done. We have now defined all elements in each figure.\n- In many of the bargraphs it is difficult to interpret the effect of the treatments. Can\nyou add a compact letter display to indicate statistically significant differences between\ntreatments? I think that this would make it much easier for readers to identify\ndifferences.\nReply2-22: Sorry for the confusion. We have now re-plotted all the figures in Main\ntext and Supplementary Information, and added a compact letter to each figure to\nindicate a statistically significant difference between each treatment.\nTables: Please indicate what the italicization of numbers represents.\nReply2-23: Done.\nFig S3: Please add the years to the figure (as in Fig S5). Please add error bars to each\npoint and indicate if any differences were statistically significant in the figure caption.\nReply2-24: Done. Thanks.\nReviewer #3 (Remarks to the Author):\nGeneral comments: The manuscript provides substantial evidence for warming and N\naddition control on the role that fine roots and arbuscular mycorrhizal fungal (AMF)\ntraits play in forming soil carbon (C). Overall, this is a well-written paper that is easy\nto read. However, there are a few significant concerns. First, the novelty of the study\ndesign needs to be better articulated in the introduction. There have been many studies\nthat have evaluated the effects of warming or N addition alone on the parameters\nmeasured in this study. This study is unique in that it includes a combination treatment\n(warming x N addition); however, the authors do not highlight this at all in the\nintroduction. The simultaneous application of warming and nitrogen (N) addition has\nbeen shown to elicit synergistic effects when in combination that are otherwise not\nobserved in single-factor treatments (warming and N alone). The authors thus miss a\ncritical opportunity to highlight the significance of their study.\nReply3-1: Thank you for your encouragement and insightful comments. We agree that\nthe novelty of our experimental design, specifically the combined warming ×N addition\ntreatment, was underemphasized in the original submission. To address this, we have\nnow added more information in the Introduction and expanded our Discussion to\nhighlight the importance of studying interactive effects, as the simultaneous application\nof warming and N addition can produce synergistic or non-additive responses that are\nnot detectable under single-factor treatments.\nAlso, we have re-analyzed our data and highlighted the results of interactions between\nwarming and N addition in our revised manuscript.\nSecond, there is a potential major flaw with the mineral soil C data. It appears that all\nsoil was dried at 65°C. However, standard methods for determining gravimetric water\ncontent use 105°C for mineral soils (65°C is used for plant materials and organic soil\nhorizons). Thus, there is likely an underestimate of soil water content and an\noverestimate of soil C. This is especially problematic in a study like this where there\nare differential treatment differences in soil moisture (i.e., warming will dry the soil\nrelative to the control and N addition plots).\nReply3-2: We agree that soil water content is often measured through drying soils\nunder 105 oC. However, heating soils under 105 oC may induce loss of some organic C\n(particularly some small molecular volatile organic C). Because we want to accurately\ndetect the soil C as much as possible, we intentionally used a lower temperature (60 oC\nor 65 oC) for this purpose, which has been proposed and used by other top scientists in\nthe related areas (e.g., Cotrufo et al. 2019, Nature Geoscience; Delgado-Baquerizo et\nal. 2023, Nature Climate Change).\nAlso, we emphasize that our study compared the C content under different treatments\non the same soil type rather than attempted to compare the absolute C contents across\ndifferent soil types. We are confident that using the water contents obtained under 65\noC did not mask the relative treatment effects in field. Moreover, we measured soil\nmoisture at both temperatures, confirming the two temperatures heating method\nreported comparable results (y = 0.989x + 0.004; R2 = 0.995; p < 0.001; see Figure\n105 65\nR8 below), and should easily convert our C results to a dry soil base obtained under\n105 oC heating.\nResponse Figure 8: Relationship between soil moisture determined under 105 oC and\n65 oC.\nThere is also an over-interpretation of the fine root and AMF trait data. The authors\nsuggest that changes in plant adaptive responses to global change influence soil C. It\nremains unclear whether the plant response was due to plasticity at the individual level\nor a plant community composition shift (which the authors report as occurring). In all\ncases, this is likely not an adaptive response and is more likely a combination of\nacclimation at the species level and a shift in community composition.\nReply3-3: Insightful comment. We agree that a combination of acclimation at the\nspecies level (e.g., reduced biomass C:N ratio under N input) and a shift in community\ncomposition better describe what happened in root traits. We have now modified the\ntext following your advice (see Line 53-56).\nRegarding the ingrowth core experiment, it needs to be clarified how root-derived C\nwas calculated. For example, because roots cannot be isolated from AMF, AMF-C\nshould be subtracted from the total C deposited in the core to get a true measure of root-\nderived C. Was this done?\nReply3-4: Insightful comment. We agree that accurately quantifying root-derived C\nrequires separating AMF-C from total C deposited in the ROOT-ingrowth cores. We\nhave now subtracted AMF-derived C from ROOT (or Root+AMF-)-derived C to get\na true measure of root-derived C, and compared with ROOT -derived C (see\nResponse Figure 9 below). Our results showed that there was an interaction of N input\nand warming on root-derived C (after AMF-C subtraction). Compared with the control,\nNr input, warming and the combination of Nr input and warming reduced root-derived\nC by 30.1%, 38.0% and 25.3%, respectively.\nHowever, AMF are obligate symbionts with the roots of most land plants, that is, they\nmust colonize plant roots to grow and reproduce (Smith & Read 2008). To our\nknowledge, nobody has so far grown AMF in soil or media independent of plants (To\nbe more accurate: On 12 Jan., 2022, Tanaka et al. has just reported that they have\ngenerated asymbiotic mass production of the arbuscular mycorrhizal fungus\nRhizophagus clarus. Communications Biology). Therefore, effects of roots and AMF\nare confounded in soil, making the quantification of AMF or root effects particularly\nchallenging. The ROOT-derived C here in our Main Text included both AMF-C and\nroot-derived C.\nResponse Figure 9: Effects of Nr input and warming on root-derived C (a) and ROOT-\nderived C (b) after 2-year incubation. Data show mean ± SE (n = 4). Significant\ndifferences as determined by two-way ANOVA (*p < 0.05 and **p < 0.01) and two-\nsided Tukey’s HSD are indicated by different letters. Con represents control; N\nrepresents N input; W represents warming only; and N+W represents combined N input\nand warming. ROOT refers to roots with AMF.\nAdditionally, there were no dissolved organic C (DOC) control cores reported. A\nsignificant portion of new C deposited into the core could be from DOC inputs via rain\nand soil water lateral flow. In my experience, warmed soils show significantly more\nDOC contamination using this method.\nReply3-5: We agree that theoretically, some dissolved organic C (DOC) may have\nmoved laterally along with water into the AMF- and/or root-ingrowth cores, which may\nlead to elevated estimates of ROOT-derived C. However, this effect is likely minimal,\nbecause lateral flows of DOC are typically minor relative to mycorrhizal/root C fluxes,\nas demonstrated by numerous previous studies (Michalzik et al. 2001; Huang et al. 2020;\nKeller et al. 2021). Also, lateral water movement in our arid system is very low. Finally,\nwe measured DOC concentrations in all soil cores, including no-AMF controls, and\nfound no significant effect of warming on DOC content (see Figure R4 above),\nsuggesting that warming-induced changes in soil moisture did not affect lateral C\nmovement (Line 506-513).\nFinally, no interaction term (warming x N addition) is reported. You need to test for\nthis interaction to appropriately determine the significance of your treatments given the\nfield design (full factorial). Also, it doesn’t appear that the statistical approach used\naccounted for the fact that measurements were made over time in the same plots (i.e.,\nrepeated measures)? See below for more detailed comments about each of these (and\nother) issues.\nReply3-6: Insightful comment. We have now re-analyzed our data to test the effect of\nNr input, warming and their interactions by considering the repeated factor year (see\nthe ANOVA Tables S3 and S5 for details).\nSpecific line comments:\nL59. Many studies have found that N addition suppresses decomposition, contributing\nto the accumulation of soil C. Changes to the N cycle need to be seriously considered\nin this study, rather than assuming that N increases decomposition or is not a driving\nfactor in warmed soils. Additionally, not all plants respond positively to N addition,\nand in some cases, plant growth itself is suppressed. A more thorough review and\ncitation of the literature on N addition impacts on plant growth and decomposition is\nwarranted. If considered through the lens of suppressed decomposition, the\ninterpretations of this study would change entirely.\nReply3-7: Thank you for this valuable comment. Yes, we agree that many studies have\nfound that Nr addition suppresses decomposition, particularly in forest ecosystems,\ncontributing to the accumulation of soil C (e.g., Fog 1988; Janssens et al. 2010; Lu et\nal. 2021). Moreover, plant responses to Nr addition vary by plant species and\necosystems, with some studies reporting neutral or even negative effects on plant\ngrowth under elevated N availability. In light of this, we have now re-written the\nIntroduction on how Nr addition affected plant growth and SOC decomposition (see\nLine 76-82 and 100-105).\nL62. A major theme of the paper is the effects of N and warming on plant dynamics.\nNitrogen availability often co-varies with soil warming. That is, warming increases N\nmineralization rates and thus N availability. This study could directly parse out the\neffects of warming versus N addition. For example, you can comment on whether\nwarming effects on the ecosystem are due to changes in water or N dynamics. The\nnovelty of the study has yet to be sufficiently conveyed.\nReply3-8: Thank you for this excellent comment. We fully agree that warming often\nincreases N mineralization rates and thus N availability, particularly in cold or water\nsufficient ecosystems (Melillo et al. 2017). Yet, our ecosystem was water-limited,\nwhere warming induced soil drying may cancel the positive effect of warming on\nmicrobes for N mineralization and plant growth. Indeed, our results showed that\nwarming alone had no significant effect on soil NH + or NO - (see Supplementary Table\n4 3\n2; Figure R10).\nImportantly, the full-factorial design of our experiment (warming × N addition) allowed\nus to separate the effects of warming from those of external N inputs. By doing so, we\nwere able to evaluate whether observed changes in plant, root and AMF traits were\nmore attributable to changes in water dynamics, nutrient supply, or their interaction,\nadding novelty and mechanistic insight to our study.\nResponse Figure 10: Effects of Nr input and warming on soil available NO - in 2018\n3\n(a), 2019 (b), 2021 (c) and over the 3 years (d), respectively. Significant differences as\ndetermined by two-way ANOVA (*p < 0.05, **p < 0.01 and ***p < 0.001) and two-\nsided Tukey’s HSD are indicated by different letters. Con represents control; N\nrepresents N input; W represents warming only; and N+W represents combined N input\nand warming.\nL67. This is relevant to mycorrhizae generally and not just AMF. Since the study\nsystem has not yet been defined, it’s important to be more general or to describe why\nyou are focusing on AMF specifically.\nReply3-9: Good point. We have now defined AMF system in the first paragraph and\nmodified the sentence to be more accurate (Line 60-62 and 100-101).\nL76. The term “priming” has a specific definition which is that labile plant C inputs\nstimulate the decomposition of native soil organic matter. Clarify the language here.\nReply3-10: Thanks. Done (Line 64).\nL80. This assumes that N addition equally affects the members of a community, which\nmay not be a valid assumption. Does adding N favor certain grasses or forbs based on\ndifferent root foraging strategies?\nReply3-11: Thanks for pointing this out. Yes, Nr input did not equally affect the\nmembers of a plant community. Indeed, we found that N favored the growth of grass\nStipa grandis over forb Carex spp by the year of 2021. This aligns with previous\nfindings that Nr input can alter plant community composition due to competitive\nexclusion of some plant species by promoting nitrophilic species (e.g., Simkin et al\n2016). Specifically, N input tends to promote the dominance of grasses over forbs in\ngrasslands, likely due to their different capacity for nutrient uptake (Stevens et al. 2006;\nLi et al. 2020). Compared with forbs, most grasses have highly branched fibrous roots\nthat are more efficient in promoting nutrient uptake (Chapin et al. 1995; Stevens et al.\n2006).\nL92. Water is not the only factor that warming influences. As noted above, there should\nbe careful consideration of changes in N cycling under warming. Can you provide\ninformation on N availability in response to the treatments? Was there any evidence of\na drought by plants? Otherwise, it is difficult to attribute the warming treatment to water\ndeficit without having a warming x drought treatment. But you can comment on\nwhether warming and N addition plots had similar effects. This will allow you to at\nleast be more confident that the effects were due to water and not N.\nReply3-12: Great suggestion. Our results showed that Nr input significantly increased\nsoil available N (mainly NO -), and warming further amplified this effect, but warming\n3\nalone had no significant effect on it (see Figure R10 above; Supplementary Tables 2\nand 3). One possible reason for the N×warming interaction on NO - was that warming\n3\nreduces soil moisture and NO - loss through leaching and denitrification.\nchanges solely to water limitation should be done cautiously. However, given that\nwarming alone did not affect soil N availability, and that warming consistently reduced\nsoil moisture (see Supplementary Figure 3), we interpret the warming effects in our\nstudy as primarily driven by reduced water availability rather than changes in N\ndynamics.\nL104-106. The first hypothesis is not a strong scientific hypothesis in that it isn’t\ndirectional (i.e., how specifically are N inputs predicted to shift the plant community?)\nand no mechanisms is provided. Having said that, I’m not in favor of writing hypotheses\nafter the data are in hand, since that then influences how the hypothesis is written.\nReply3-13: Thanks. We have now revised our Introduction section to introduce how\nNr input affected plant community composition to better articulate our scientific\nhypothesis (see Line 100-105 and 139-141).\nL106. The phrase “priming residue decomposition” is imprecise and unclear. Although\nI understand what you mean, someone unfamiliar with the priming process may not.\nReply3-14: Thanks for pointing this out. We have now revised “priming residue\ndecomposition” as “priming microbial growth and enzyme production, and thereby\nstimulating soil organic C decomposition” (see Line 140-141).\nL130. Can you tell whether this was due to the change in plant community? For those\nunfamiliar with this ecosystem, do the new species produce more biomass or have\ninvasive-like qualities?\nReply3-15: Yes, our results showed that Nr input altered plant community favoring the\ngrowth of grasses dominated by Stipa grandis over forbs dominated by Carex spp. Also,\ncompared with forbs, grasses often have highly branched fibrous roots and are more\nefficient in nutrient forging (Stevens et al. 2006).\nAlthough Stipa grandis is not considered invasive in our semi-arid grassland ecosystem,\nit tends to become dominant under elevated N availability due to its competitive\nadvantage. This shift may lead to reduced plant diversity and increased aboveground\nbiomass relative to forb-dominated communities.\nL139. Root turnover in your study was primarily a function of root biomass, not\nproduction. i.e., turnover was driven by standing biomass, and I am skeptical of how\naccurately this reflects true turnover.\nReply3-16: Sorry for the confusion. Root turnover was calculated following the widely\naccepted method outlined by Gill & Jackson (2000):\nFine root turnover (yr-1) = annual belowground production / maximum standing\nbiomass\nBriefly, we installed root-ingrowth core with a mesh size of 1.8 mm into each field plot\nto allow fine roots to grow into the soil and estimate the newly grown root as root\nproduction over the growing season. Standing biomass was determined using soil cores\nat peak biomass of plant vegetation. Also, this method has been used in our previous\npublication (Zhang et al. 2023) and other studies (Lukac et al. 2003; McCormack et al.\n2014; Li X et al. 2021).\nL200. I understand why this is called AMF-derived C, but it may also include other\nfungi.\nReply3-17: Yes, we agree that it may also include other fungi (see more explanation in\nReply2-5). We have now modified the sentence to clarify why this is called AMF-\nderived C.\nL225. Clarify whether these were traits measured from bulk soil or soil from the\ningrowth core.\nReply3-18: Sorry for the confusion. These traits were measured from ingrowth core\nsoil. We have clarified this in the Method (see Line 416-417).\nL251. Terms like fragile are vague. Is there a more quantitative way to describe roots\nthat turnover faster?\nReply3-19: We have now used mortality or short lifespans to replace fragile (Line 295\nand 317).\nL255. You can’t be certain that this would increase root exudate inputs. I would instead\nchange language in the text to generally say “may increase root-derived C”\nReply3-20: Good comment. We have now modified the sentence (Line 315-318).\nL256. Counterintuitive. Earlier, the paper stated that the plants and microbes were N\nand P limited. This needs to be clarified.\nReply3-21: Sorry for the confusion. We have now modified this sentence. “As soil\nsaprophytic microbes are generally more C-limited rather than N and P-limited in\ntemperate grasslands” (Line 320-321).\nL366. How many root tips were counted? Often hundreds of tips need to be counted to\ndetermine the colonization rate successfully. Clarify.\nReply3-22: Each dried root sample was cut into 100 segments with the length of\napproximately 1 cm to determine mycorrhizal colonization rate (see Line 427-435 for\ndetails).\nL423. How were DOC inputs determined?\nReply3-23: After we collected all ingrowth-chambers in field back, soil DOC was\ndetermined from 20 g soils (dry soil equivalent) extracted with 50 mL of 0.5 M K SO\n2 4\nafter shaking for 30 min. We have now added the information in the Method (Line 402-\n405).\nL452 Clarify if AMF-C was subtracted from root C recovered in cores. If not, you\nshould change the language to “root + fungal-derived C“, since both roots and hyphae\ngrew into the cores.\nReply3-24: Good point. We have made all the changes. Please see our more\nexplanations in Reply3-4.\nL461. Is this a standard method?\nReply3-25: Well, we would say it is a commonly used method. Using this isotope\nmethod, we have published mycorrhizae-related research in high-impact journals\n(Cheng et al. 2012; Qiu et al. 2021). Also, other researchers have used the method in\nthe related areas (see Verbruggen et al. 2018; Zhang et al. 2020).\nL483. It is standard protocol to dry mineral soils at 105°C to remove soil pore water,\nthus soil moisture content is very likely underestimated which then influences soil C\ncontent (likely an overestimate). Since warming influences soil moisture, this could\ninfluence the relative differences between treatments.\nReply3-26: Good point. We agree that it is a standard protocol to dry mineral soils at\n105 °C to remove soil pore water. However, heating soils under 105 oC may induce loss\nof some organic C (particularly some small molecular volatile organic C) (please see\nour more explanations in Reply3-2).\nL502. Was the interaction between warming and N addition tested? It is not reported in\nthe methods, nor does this show in any figure.\nReply3-27: Thanks for pointing this out. We have now reported the interaction between\nwarming and N addition if there was in the figures and in the Supplementary Tables.\nAlso, we have revised our results to better interpret how N input and warming interacted\nto affect roots and their associated mycorrhizal fungi and their resulting impact on SOC\ndynamics.\nL502. I’m concerned about the importance of time in this study since you make repeated\nmeasures in the same plots. Your model did not include time, which could be a\nsignificant factor in structuring the root traits and soil C patterns. For example, did each\nyear have similar weather patterns? Was the heating consistent each year? Other\nenvironmental variables may vary over time, and thus time would be an important\nfactor to account for this variability. It would also be good to know in which year the\nbiggest changes occurred. If time is insignificant in your model, you may proceed with\nANOVA as you have conducted them, but the effect of time should be noted.\nReply3-28: Great comment. We have now conducted repeated measures ANOVA to\nconsider time effect on root traits and AMF community composition and added the\nresults in Supplementary table.\nEnvironmental conditions indeed varied across years. For example, mean annual\nprecipitation at the study site was abnormally high at ca. 639 mm in 2018 but decreased\nto ca. 569 mm in 2019 and ca. 462 mm in 2021 (Zhang et al. 2023). Thus, time can be\nan important factor to account for the variability. For example, we found that fine roots\nwere significantly thinner and longer in drier year (2021) than that in wet year (2018)\n(see Supplementary Figure 6).\nIt needs to note that our warming treatment consistently increased air temperature by\n2.0°C, 2.6°C, and 2.1°C, and reduced average soil volumetric moisture by 15.1%,\n16.8%, and 13.5% in 2018, 2019, and 2021, respectively. Despite these variations, the\nwarming effects on root traits and AMF remained consistent between different years\n(see Supplementary Table 5 and 6).\nL739. There is a lot of repetition in the figure. One suggestion would be to keep the\nmatrix and the PCA and move the individual variables that comprise the PCA to a\nsupplemental table.\nReply3-29: Good suggestion. We have added the PCA result in the Table S10.\nTable S10. The results of the principal component analysis of root traits base on raw\ndata from 2018, 2019 and 2021 as shown in Fig. 2. Displayed is the Eigenvalue as well\nas the proportion of variance explained by each principal component (PC) and loadings\nof the root traits.\nPC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8\nEigenvalue 3.240 2.525 1.252 0.641 0.235 0.075 0.017 0.014\nVariance 40.503 31.566 15.649 8.013 2.934 0.941 0.212 0.181\nAMF (%) -0.476 -0.028 0.264 0.188 -0.813 -0.066 -0.0486 0.013\nRTD -0.179 0.445 -0.437 -0.458 -0.146 -0.434 0.399 -0.004\nSRL 0.542 -0.027 -0.000 -0.017 -0.240 -0.620 -0.510 -0.052\nD -0.456 -0.235 0.231 0.258 0.454 -0.633 0.082 -0.059\nSRSA 0.478 -0.247 0.223 0.199 -0.187 -0.128 0.755 0.024\nRNC -0.072 -0.568 -0.034 -0.500 -0.079 0.051 0.011 -0.642\nRCC 0.028 0.176 0.746 -0.585 0.090 -0.012 -0.014 0.246\nRCN 0.080 0.575 0.280 0.238 0.062 0.044 0.038 -0.721\nL758. Is there a significant interaction between warming and N addition?\nReply3-30: There was ONLY a significant interaction between warming and N\naddition on ROOT-derived C. We have now added different lowercase letters in Fig. 4\nto indicate the difference among different treatments.\nReferences:\n1. Chapin, F., Shaver, G., Giblin, A., Nadelhoffer, K. & Laundre, J. Responses of arctic tundra to\nexperimental and observed changes in climate. Ecology 76, 694–711 (1995).\n2. Chen, Z. et al. Effects of plant diversity on semiarid grassland stability depends on functional\ngroup composition and dynamics under N and P addition. Sci. Total Environ. 799, 149482\n(2021).\n3. Cheng, L. et al. Arbuscular mycorrhizal fungi increase organic carbon decomposition under\nelevated CO . Science 337, 1084–1087 (2012).\n2\n4. Cotrufo, M. F., Ranalli, M. G., Haddix, M. L., Six, J. & Lugato, E. Soil carbon storage informed\nby particulate and mineral-associated organic matter. Nat. Geosci. 12, 989–994 (2019).\n5. Davidson, E. A. & Janssens, I. A. Temperature sensitivity of soil carbon decomposition and\nfeedbacks to climate change. Nature 440, 165–173 (2006).\n6. Delgado-Baquerizo, M. et al. Biogenic factors explain soil carbon in paired urban and natural\necosystems worldwide. Nat. Clim. Change 13, 450–455 (2023).\n7. Faust, S. et al. Effect of biodynamic soil amendments on microbial communities in comparison\nwith inorganic fertilization. Appl. Soil Ecol. 114, 82–89 (2017).\n8. Fog, K. The effect of added nitrogen on the rate of decomposition of organic matter. Biol. Rev.\n63, 433–462 (1988).\n9. Gill, R. A. & Jackson, R. B. Global patterns of root turnover for terrestrial ecosystems. New\nPhytol. 147, 13–31 (2000).\n10. Han, Y., Feng, J., Han, M. & Zhu, B. Responses of arbuscular mycorrhizal fungi to nitrogen\naddition: A meta-analysis. Glob. Change Biol. 26, 7229–7241 (2020).\n11. Hu, H. et al. Responses of AM fungal abundance to the drivers of global climate change: A\nmeta-analysis. Sci. Total Environ. 805, 150362 (2022).\n12. Huang, J. et al. Plant carbon inputs through shoot, root, and mycorrhizal pathways affect soil\norganic carbon turnover differently. Soil Biol. Biochem. 160, 108322 (2021).\n13. Irving, T. B., Alptekin, B., Kleven, B. & Ane J. M. A critical review of 25 years of glomalin\nresearch: a better mechanical understanding and robust quantification techniques are required.\nNew Phytol. 232, 1572–1581 (2021).\n14. Jach-Smith, L. C. & Jackson, R. D. N addition undermines N supplied by arbuscular\nmycorrhizal fungi to native perennial grasses. Soil Biol. Biochem. 116, 148–157 (2018).\n15. Janssens, I. A. Et al. Reduction of forest soil respiration in response to nitrogen deposition.\nNature Geosci. 3, 315–322 (2010).\n16. Jilling, A. et al. Minerals in the rhizosphere: over looked mediators of soil nitrogen availability\nto plants and microbes. Biogeochemistry 139, 103–122 (2018).\n17. Keiluweit, M. et al. Mineral protection of soil carbon counteracted by root exudates. Nat. Clim.\nChange 5, 588–595 (2015).\n18. Keller, A. B. et al. Root-derived inputs are major contributors to soil carbon in temperate forests,\nbut vary by mycorrhizal type. Ecol. Lett. 24, 626–635 (2021).\n19. Li, W. et al. The effects of simulated nitrogen deposition on plant root traits: A meta-analysis.\nSoil Bio. Biochem. 82, 112–118 (2015).\n20. Li, X. et al. Canopy and understory nitrogen addition have different effects on fine root\ndynamics in a temperate forest: implications for soil carbon storage. New Phytol. 231, 1377–\n1386 (2021).\n21. Li, S. et al. Mowing alters nitrogen effects on the community-level plant stoichiometry through\nshifting plant functional groups in a semi-arid grassland. Environ. Res. Lett. 15, 074031 (2020).\n22. Li, Z. et al. Climate change drivers alter root controls over litter decomposition in a semi-arid\ngrassland. Soil Biol. Biochem. 158, 108278 (2021).\n23. Lu, X., Vitousek, P. M., Mao, Q., Gilliam, F. S. & Mo, J. Nitrogen deposition accelerates soil\ncarbon sequestration in tropical forests. Proc. Natl. Acad. Sci. USA 118, e2020790118 (2021).\n24. Lukac, M., Calfapietra, C. & Godbold, D. L. Production, turnover and mycorrhizal colonization\nof root systems of three Populus species grown under elevated CO (POPFACE). Glob. Change\n25. McCormack, L., Adams, T., Smithwick, E.A.H. & Eissenstat, D. M. Variability in root\nproduction, phenology, and turnover rate among 12 temperate tree species. Ecology 95, 2224–\n2235 (2014).\n26. Melillo, J. M. et al. Long-term pattern and magnitude of soil carbon feedback to the climate\nsystem in a warming world. Science 358, 101–105 (2017).\n27. Michalzik, B. et al. Modelling the production and transport of dissolved organic carbon in forest\nsoils. Biogeochemistry 66, 241–264 (2003).\n28. Ngosong, C., Gabriel, E. & Ruess, L. Use of the Signature Fatty Acid 16:1ω5 as a Tool to\nDetermine the Distribution of Arbuscular Mycorrhizal Fungi in Soil. J Lipids. 2012, 236807\n(2012).\n29. Qiu, Y., et al. Warming and elevated ozone induce tradeoffs between fine roots and mycorrhizal\nfungi and stimulate organic carbon decomposition. Sci. Adv. 7, eabe9256 (2021).\n30. Rodríguez, A., et al. Fairy ring-induced soil potassium depletion gradients reshape microbial\ncommunity composition in a montane grassland. Eur. J. Soil Sci. 73, 1–15 (2022).\n31. Simkin, S. M. et al. Conditional vulnerability of plant diversity to atmospheric nitrogen\ndeposition across the United States. Proc. Natl. Acad. Sci. USA 113, 4086–4091 (2016).\n32. Smith, J. L. & Paul, E. A. in Soil Biochemistry Vol. 6 (eds Bollag, J. & Stotzky, G.) 357– 395\n(Marcel Dekker, New York, 1990).\n33. Stevens, C. J., Dise, N. B., Gowing, D. J. & Mountford, J. O. Loss of forb diversity in relation\nto nitrogen deposition in the UK: regional trends and potential controls. Glob. Change Biol. 12,\n1823–1833 (2006).\n34. Tanaka, S. et al. Asymbiotic mass production of the arbuscular mycorrhizal fungus\nRhizophagus clarus. Commun. Biol. 5, 43 (2022).\n35. Verbruggen, E. J., Jansa, J., Hammer, E. C., Rillig, M. C. & Vries, F. Do arbuscular mycorrhizal\nfungi stabilize litter-derived carbon in soil? J. Ecol. 104, 261–269 (2016).\n36. von Lützow, M. et al. Stabilization of organic matter in temperate soils: mechanisms and their\nrelevance under different soil conditions—a review. Eur. J. Soil Sci. 57, 426–445 (2006).\n37. Walder, F. et al. Tracking the carbon source of arbuscular mycorrhizal fungi colonizing C3 and\nC4 plants using carbon isotope ratios (δ13C). Soil. Biol. Biochem. 58, 341-344 (2013).\n38. Wallander, H., Nilsson, L. O., Hagerberg, D. & Baath, E. Estimation of the biomass and\nseasonal growth of external mycelium of ectomycorrhizal fungi in the field. New Phytol. 151,\n753–760 (2001).\n39. Wang, J. et al. Fine root functional trait responses to experimental warming: A global meta-\nanalysis. New Phytol. 230, 1856–1867 (2021).\n40. Wei, L. et al. Nitrogen addition decreased soil respiration and its components in a long-term\nfenced grassland on the Loess Plateau J. Arid Environ. 152, 37–44 (2018).\n41. Zhang, J., Ekblad, A., Sigurdsson, B. D. & Wallander, H. The influence of soil warming on\norganic carbon sequestration of arbuscular mycorrhizal fungi in a sub-arctic grassland. Soil Biol.\nBiochem. 147, 107826 (2020).\n42. Zhang, K. et al. Moderate precipitation reduction enhances nitrogen cycling and soil nitrous\noxide emissions in a semi-arid grassland. Glob. Change Biol. 29, 3114–3129 (2023).\n43. Zhang, Z., Phillips, R. P., Zhao, W., Yuan, Y., Liu, Q. & Yin, H. Mycelia-derived C contributes\nmore to nitrogen cycling than root-derived C in ectomycorrhizal alpine forests. Funct. Ecol. 33,\n346–59 (2019).\nPoint by point responses to Reviewers' Comments on Manuscript\nNature Communications (MS# NCOMMS-23-28603B)\n(Comments in black and Responses in blue)\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe MS titled ” Root traits and mycorrhizal fungal community mediate reactive N and\nwarming impacts on soil organic carbon” provides substantial insight into\nunderstanding of soil C stabilization as affected by climate change. As I have had a\npleasure to review this MS before, I will provide my review in relation to my earlier\ncomments and answers to them. The MS is improved; however, I still have some\ncomments.\nReply1-1: We sincerely thank you for your support to our manuscript. We have\ncarefully revised the manuscript following your comments and suggestions, and believe\nthat all concerns have now been adequately addressed.\nMy comments to abstract:\nline 42. the role of AMF in C stabilization could be omitted from 1st sentence.\nReply1-2: Good suggestion. We have now omitted the mention of “the role of AMF in\nC stabilization” from the sentence (Line 40-41).\nline 53 – “C-protecting Glomeraceae” – this is unclear.\nReply1-3: The new revision has removed this term and revised the related sentence to\n“Nr input also increased soil N:P ratios and shifted AMF communities toward taxa\nwith finer extraradical hyphae, weakening SOC protection”, to more clearly and\naccurately convey its intended meaning (see Line 48-49).\nIntroduction:\nline 61 – Authors stated that SOC originates particularly from AMF. I cannot see good\nconfirmation for it in cited papers (3 and 4). Could you maybe rephrase?\nReply1-4: Thank you for pointing this out. We agree that the original wording may\nhave overstated the contribution of AMF to SOC origin. We have now rephased the\nsentence and replaced the reference (3) with Bai et al., 2022 and Liang et al., 2020,\nwhich more directly support the revised the statement.\n“Over 80% of SOC originates from roots and root-associated microbes3-5, with\narbuscular mycorrhizal fungi (AMF) representing an important component of these\nbelowground inputs.” (Line 55-57).\nReferences:\nBai, Y. & Cotrufo, M. F. Grassland soil carbon sequestration: Current understanding,\nchallenges, and solutions. Science. 377, 603–608 (2022).\nLiang, C., Amelung, W., Lehmann, J. & Kästner, M. Quantitative assessment of\nmicrobial necromass contribution to soil organic matter. Glob. Change Biol. 25, 3578–\n3590 (2019).\nline 78 - maybe “accrual” instead of “accruals”\nReply1-5: Corrected.\nline 81 – Authors refer to metanalysis from 2005, I would suggest to read more up to\ndate metaanalyses for example 10.1111/gcb.16588, which provide new angle on the\nmechanism behind the effect of N addition on SOC. I would also suggest refreshing the\nliterature, there is no paper from 2024 and 2025!\nReply1-6: Thank you for this helpful suggestion. We agree that relying on an early\nmeta-analysis may overlook more recent mechanistic insights. We have therefore\nupdated the Introduction by incorporating the meta-analysis of Tang et al., (2023)\n(Global Change Biology; 10.1111/gcb.16588), which provides a more mechanistic\nperspective on how N addition influences SOC dynamics. In addition, we have\nrefreshed the literature by including several relevant studies published in 2024\n(Machmuller et al., 2024) and 2025 (Seabloom et al., 2025; Tang et al., 2025), ensuring\nthat the manuscript reflects the most up-to-date understanding of N effects on SOC.\nReferences:\nMachmuller, M. B. et al. Arctic soil carbon trajectories shaped by plant–microbe\ninteractions. Nat. Clim. Chang. 14, 1178–1185 (2024).\nSeabloom, E. W., Hobbie, S. E., MacDougall, A. S. & Borer, E. T. Multidecadal\npersistence of soil carbon gains on retired cropland following fertilizer cessation. Nat.\nGeosci. 18, 1014–1019 (2025).\nTang, B., Rocci, K. S., Lehmann, A. & Rillig, M. C. Nitrogen increases soil organic\ncarbon accrual and alters its functionality. Glob. Change Biol. 29, 1971–1983 (2023).\nTang, S. et al. Soil carbon sequestration enhanced by long-term nitrogen and\nphosphorus fertilization. Nat. Geosci. 18, 1005–1013 (2025).\nline 84 – please add “factor” after “limiting”\nReply1-7: Done.\nline 85 – I cannot agree with this statement. The amount of papers in the topic has\ndramatically increased in last years, thus I do not think that soil is treated as “black box”\nanymore, and that roots are neglected. Moreover, statement that roots are neglected\n(line 87) is in contradiction with line 90-91, where Authors stated that root traits are\nkey drivers.\nReply1-8: Insightful comment. We agree that our original statement does not\naccurately reflect the current state of the field, which has seen a significant growth in\nresearch in recent years. The phrasing has been revised to address both points raised:\n“While the individual impacts of Nr input and warming on SOC dynamics have been\nextensively studied9,16, the roles of roots and AMF in mediating SOC responses to\ncombined Nr and warming influence have received comparatively less attention.” (Line\n80-83).\nline 94 – what is the role of dot at the end of line 94?\nReply1-9: Sorry for this typo. We have deleted the dot.\nline 95 – is this sentence necessary?\nReply1-10: Good point. The sentence has been deleted and modified as suggested (Line\n91-93).\nline 99 – “.. impact of AMF on SOC dynamics is unexplored” – here is an example of\npaper in such topic: 10.1111/nph.18914. Please check for newest papers in this field.\nThis comment refers also to line 131.\nReply1-11: We appreciate the reviewer’s comment and the provided reference. We\nhave updated the text at lines 99 and 131 to reflect recent advancements in the field (He\net al., 2025; Liu et al., 2025), and have cited relevant recent literature, including the\npaper suggested (Horsch et al., 2023; 10.1111/nph.18914).\nReferences:\nHe, T. et al. Precipitation increase promotes soil organic carbon formation and stability\nvia the mycorrhizal fungal pathway. Proc. Natl. Acad. Sci. USA. 122, e1775895174\n(2025).\nHorsch, C. C. A., Antunes, P. M., Fahey, C., Grandy, A. S. & Kallenbach, C. M. Trait-\nbased assembly of arbuscular mycorrhizal fungal communities determines soil carbon\nformation and retention. New Phytol. 239, 311–324 (2023).\nLiu., C. et al. Arbuscular mycorrhizal fungi hyphal density rather than diversity\nstimulates microbial necromass accumulation after long-term Robinia pseudoacacia\nplantations. Soil Biol. Biochem. 206, 109817 (2025).\nline 143 – though Authors added microbial necromass measurements, they still refer\nonly to MAOC as pool of stable C. I would suggest reevaluating hypotheses in line with\nadded analyses.\nReply1-12: Thank you for pointing this out. We have revised our hypotheses to include\nboth MAOC and microbial necromass as pools of stable C.\nResults and Discussion:\nAs the MS provides a lot of data, I would suggest dividing them. Otherwise, the text\nreads more like a results section mostly. This very descriptive style ends with\nconclusion-like paragraph (line 344-351) with very overall statements. I would\nrecommend separation of Discussion.\nReply1-13: Thank you for your suggestion. We do agree that a separation of Discussion\nmay improve the clarity. However, keeping the Results and Discussion together\nimproves logic flow. While we did not divide the Results and Discussion into two\nindependent sections, we substantially revised the Results and Discussion by reducing\ndescriptive content and strengthening interpretation, as you advised.\nline 270 and other lines – why root is written as ROOT?\nReply1-14: We apologize for the lack of clarity. We have revised the text to explicitly\nstate that \"ROOT\" refers to \"the penetration of both roots with AMF\", when it first\nappeared in the Main text (see Line 246-247).\nline 541 – this protocol measures also mannosamine.\nReply1-15: Yes. This analytical protocol also measures manosamine. We have revised\nthe sentence at line 541 to explicitly state that our measurement of amino sugars\nincluded glucosamine, galactosamine, mannosamine and muramic acid (Line 561-563).\nAdditional comment about added analyses: As I suggested, authors added microbial\nnecromass analysis. I proposed also to add glomalin (produced by AMF) and here some\nmisunderstanding came. My point of measurement of glomalin was not to extrapolate\nit into AMF biomass, but to estimate C stabilization as affected by AMF.\nReply1-16: Thank you for the clarification regarding the purpose of measuring\nglomalin. We understand the intent was to assess C stabilization. As noted, we did not\nmeasure glomalin due to ongoing methodological debates about its nature and\nquantification. Therefore, to assess the stabilization of microbial-derived organic matter,\nwe quantified amino sugar-derived microbial necromass C as a direct measure of\nmicrobial residues, and MAOC as indicators of organo-mineral associated C pool\n(including Fe- and Ca-associated OC). These are well-established and complementary\nmetrics for assessing stabilized microbial-derived organic matter, which directly\naddress our core aim of evaluating the pathways of SOC persistence under the\nexperimental treatments.\nReviewer #2 (Remarks to the Author):\nThank you for your effort--the revisions have addressed most of my previous comments\nand improved the manuscript. I think that the manuscript can make a valuable addition\nto the literature, but I am still struggling to understand some of the main findings. Upon\nreading the revised manuscript, I think that this is partly due to the claims made\nregarding the relationship between AMF and SOC dynamics (including both\ndescriptions of what has been demonstrated by existing publications and what is shown\nin this study). To me, these claims seem a bit overstated and imprecise, sometimes citing\nliterature incorrectly or overinterpreting the results. See specific comments below for\nsome examples. The manuscript would be improved by a revised interpretation of these\nfindings and their implications.\nReply2-1: Thank you very much for your positive comments and encouragement. We\nhave carefully revised the manuscript according to your valuable suggestions and\ncomments to further improve the clarity and presentation.\n52-53, 260-262, 285-287: The interpretation of the effects of AMF on SOC is somewhat\nunclear and contradictory. E.g., on lines 52-53 Glomeraceae is described as “C-\nprotecting” compared to Paraglomeraceae and Gigasporaceae, even though some of the\nsoil C pools decreased in the presence of AMF under disturbance treatments. Perhaps I\ndon’t understand the results, but even if relative abundance of Glomeraceae compared\nto other AMF is positively correlated with C pools (Figs S13 & S14), it seems\nmisleading to call it “protective” if Glomeraceae growth into the cores still results in a\nnet decrease (e.g., in some of the C pools shown in Figs 5 and S12). Or are you\nsuggesting that only the Paraglomeraceae and Gigasporaceae were responsible for C\nloss? Please clarify.\nReply2-2: Thank you for this important critique. We agree that labelling Glomeraceae\nas “C-protecting” was an overstatement. Our data show associations, not definitive\nmechanisms. We have revised the abstract to present a more accurate and nuanced\ninterpretation: “Nr input also increased soil N:P ratios and shifted AMF communities\ntoward taxa with finer extraradical hyphae, weakening SOC protection.” (Line 48-49).\nWe have also expanded the interpretation in the discussion to clarify: “The positive\ncorrelations between Glomeraceae abundance and AMF-derived C or MAOC, but the\nnegative correlations between Paraglomeraceae and/or Gigasporaceae abundance\nand AMF-derived C or MAOC (Supplementary Fig. 14) provide indirect evidence that\ndifferent AMF lineages have distinct functional impacts on soil C dynamics, and that\nthe observed community shift toward taxa with finer and less dense hyphae may reduce\nAMF capacity in enmeshing soil aggregates and protecting organic C.” (Line 354-360).\n53-56: The evidence is more correlational than mechanistic. Please revise.\nReply2-3: Good comment. We have revised the sentence.\n“Together, these findings highlight root–AMF interactions as critical regulators and\nimprove predictions of long-term SOC dynamics under future climate change.” (Line\n49-51).\n128-131: Based on my reading of Horsch et al, they did not show that AMF with thicker\nhyphae produce compounds that are conducive to the formation of MAOC, nor that this\nthereby increased SOC stability. Please revise or add references that support each of\nthese claims.\nReply2-4: Insightful comment. We have added two new references (Lovelock et al.,\n2004; Liu et al., 2025) to support our claim.\nReference:\nLovelock, C. E., Wright, S. F. & Nichols, K. A. Using glomalin as an indicator for\narbuscular mycorrhizal hyphal growth: an example from a tropical rain forest soil. Soil\nBiol. Biochem. 36, 1009–1012 (2004).\nLiu., C. et al. Arbuscular mycorrhizal fungi hyphal density rather than diversity\nstimulates microbial necromass accumulation after long-term Robinia pseudoacacia\nplantations. Soil Biol. Biochem. 206, 109817 (2025).\n128, 140, 277, 281, 287, 322: The term ‘priming’ is used imprecisely throughout the\nmanuscript and may lead to confusion. I recommend defining the term specifically and\nusing it consistently throughout the manuscript. In my understanding, priming refers to\nthe effect of new C inputs on the decomposition of existing SOC. Priming can be\n“positive” (if it leads to an increase in decomposition of existing SOC) or “negative”\n(if it leads to a decrease in decomposition of existing SOC). If you are using the term\n“priming” to refer to something else (e.g., an increase in microbial activity on line 287),\nit may be clearer to replace the term with another word that doesn’t have specific (and\nsomewhat contentious) connotations.\nReply2-5: Thanks. We have now revised the manuscript to use the term “priming effect”\nstrictly in the context of new inputs affecting the decomposition of existing soil organic\ncarbon (SOC). Where we previously used “priming” to refer to a general stimulation of\nmicrobial activity or decomposition, we have replaced it with more accurate terms such\nas “stimulated”, “accelerated”, or “increased decomposition”.\n191-194: This sentence contains some grammatical errors and is a bit difficult to follow.\nPlease revise.\nReply2-6: Sorry for the confusion. We have revised the sentence.\n“These tradeoffs in root morphology reflect a fundamental shift in plant C allocation\nstrategy mediated by changes in community composition. Specifically, Nr input\npromotes communities that invest in long, low-density roots, while warming favors\ncommunities that construct thicker, shorter roots.” (Line 191-194).\n301-308: These statements seem contradictory—did plants optimize C allocation by\nincreasing root length and surface area (to acquire resources themselves) or by investing\nmore C into AMF species that supposedly demand more C (but also provide more\nnutrients to their hosts)? Please clarify.\nReply2-7: This is an excellent and insightful question that gets to the heart of how\nplants dynamically regulate their C allocation.\nThe statements are not contradictory but\nrather describe two complementary and sequential aspects of a plant's integrated\nforaging strategy in response to changing environmental conditions.\nIn summary: The plant first reduces its overall investment in AMF as N becomes\nreadily available to enhance aboveground biomass, but then re-allocates its remaining,\nsmaller C investment to a more efficient type of AMF as other resources such as P\nbecomes limiting.\n308-310: This is an overstatement. \"C exchange and turnover at the root-soil interface”\nimplies that measurements were collected at very fine spatiotemporal resolution. Please\nrevise.\nReply2-8: Thanks for pointing this out. We have now revised the sentence:\n“Together, the observed alterations in root traits and AMF communities under Nr input\nand warming directly affect the C exchange and turnover at the root-soil interface and\ncascade to affect soil C dynamics.” (Line 323-325).\n335-336: Based on my reading of these publications, the authors did not show that AMF\nspecies differ in their capacity to generate secondary metabolites that contribute to\nMAOC. Please revise or add references that support each of these claims.\nReply2-9: Thanks for pointing this out. We should have carefully checked these\npublications to make sure they showed that AMF species differ in their capacity to\ngenerate secondary metabolites that contribute to MAOC (Lovelock et al., 2004; Liu et\nal., 2025). We have now added new references to support our statement. Also, AMF\nhyphae protect SOC by directly enmeshing soil microaggregates into macroaggregates\nand finer hyphae compromise their binding capacity.\nReferences:\nLovelock, C. E., Wright, S. F. & Nichols, K. A. Using glomalin as an indicator for\narbuscular mycorrhizal hyphal growth: an example from a tropical rain forest soil. Soil\nBiol. Biochem. 36, 1009–1012 (2004).\nLiu., C. et al. Arbuscular mycorrhizal fungi hyphal density rather than diversity\nstimulates microbial necromass accumulation after long-term Robinia pseudoacacia\nplantations. Soil Biol. Biochem. 206, 109817 (2025).\n336-338: What is evidence is there for this statement? Based on my reading of Qiu et\nal, they did not measure glomalin or its effects on soil microaggregates.\nReply2-10: Insightful comment. We have now replaced a new reference (Lovelock et\nal., 2004), which measured glomalin to better to support this statement.\nReference:\nLovelock, C. E., Wright, S. F. & Nichols, K. A. Using glomalin as an indicator for\narbuscular mycorrhizal hyphal growth: an example from a tropical rain forest soil. Soil\nBiol. Biochem. 36, 1009-1012 (2004).\n343: There is something missing from this sentence. Please revise.\nReply2-11: Thanks for pointing this out. Corrected.\n“The positive correlations between Glomeraceae abundance and AMF-derived C or\nMAOC, but the negative correlations between Paraglomeraceae and/or Gigasporaceae\nabundance and AMF-derived C or MAOC (Supplementary Fig. 13) provide indirect\nevidence that different AMF lineages have distinct functional impacts on soil C\ndynamics.” (Line 354-358).\n346-349: These claims are overstated. Please revise.\nReply2-12: Agreed. We have revised the sentences to avoid overstating our findings of\nthis study.\n“These tradeoffs enhance root and AMF turnovers and C availability for saprophytic\nmicrobes, but may reduce root- and AMF-mediated protection of soil organic\ncompounds. This could promote SOC turnover and thus compromise the potential of\nsoil C sequestration. These findings offer insights into plausible mechanisms\nunderlying the impact of reactive N and warming on plant-microbial interactions and\nSOC dynamics, which is necessary for improving predictions of soil C balance and\nterrestrial C-climate feedbacks under future climate change scenarios.” (Line 363-368).\n538-552: Galactosamine does not appear to be included in the calculation. Was it\naccounted for?\nReply2-13: Thanks for pointing this out. Yes, galactosamine was not included in the\ncalculation of microbial necromass carbon. The microbial necromass C was estimated\nusing only glucosamine (for fungal residue) and muramic acid (for bacterial residue) as\nthe standard biomarkers. We have revised the Methods section to explicitly state that\nonly glucosamine and muramic acid were used in the calculation.\nFig 6: Thank you for adding a conceptual figure. I think that in order to be impactful, it\nrequires some revision and explanation. I have several questions and comments: If\ndifferent root traits and AMF community members increase or decrease under each\nscenario, it’s confusing to list all of the them together under the titles “fine root” and\n“AMF.” Can you clarify within the figure or in the caption or text? Does N:P ratio\nchange under either scenario? If so, how? (Or perhaps this could be removed from the\nfigure?) Do the arrows coming from each plant suggest that this plant type is primarily\nresponsible for the downstream changes and C pools? If not, please change the figure\nto reflect this. Does the figure suggest that microbial necromass has a positive effect on\nAMF and root derived C? And that hyphal & root turnover has a positive effect on\norganic C decomposition? If so, please explain these relationships. Additionally, I think\nthat the word “sequestration” should be removed.\nReply2-14: Thank you for these insightful and constructive comments. We have\nsubstantially revised Figure 6 and its legend to improve clarity, interpretability, and\nconceptual accuracy. In particular, we have now 1) demonstrated how AMF and root\ntraits changed, 2) showed how N:P ratio changed under each scenario, and 3) revised\nthe arrows from plants to clearly demonstrate the plant community effect. Also, we\nrevised the figure legend to explain that microbial necromass had a positive effect AMF\nand ROOT-derived C, and hyphal & root turnover promotes a positive effect on organic\nC decomposition.\nReviewer #5 (Remarks to the Author):\nThis paper presents a study on the effects of nitrogen enrichment and warming on root\ntraits, mycorrhizal dynamics, and soil carbon. I found the paper to be well written and\ninteresting, and the authors present a wealth of important data. With some further\nrevisions it will make a valuable addition to the literature.\nReply 5-1: Thank you for your supports and insightful comments. We are very pleased\nthat you found the paper well-written, interesting, and the data important. We have\ncarefully revised the manuscript following all of your suggestions.\nI have two principal concerns with the paper:\n1) As I indicate in the line comments below, I found multiple discrepancies between the\nresults as written in the text and as presented in the figures/table/supplement. Based on\nthe response to the reviewers, it seems that this revised version of the paper includes\nupdated statistics with a Nr by warming interaction that was not included in the first\nversion of the paper. I wonder if some of the discrepancies I point out below are due to\nthe statistical tables being updated since the text was written. Although I point out some\ndiscrepancies, my list is not exhaustive and the text, figures/tables, and supplement need\nto be carefully checked for errors.\nReply 5-2: We sincerely apologize for these discrepancies. You are correct that updated\nstatistical models, including the Nr × warming interaction term, were run during\nrevision. It is possible that some text descriptions were not fully synchronized with the\nfinal statistical outputs. We have now performed a thorough, line-by-line verification\nof all results in the text against the figures, tables, and supplementary materials to\nensure complete consistency. We have also double-checked all statistical tables.\n2) Throughout the Results and Discussion section, the significance of the Nr and\nwarming treatments is discussed according to the ANOVA results. However, the figures\nshow both the ANOVA and the Tukey HSD results, and the Tukey HSD often found no\nsignificance where the ANOVA did indicate significance. This is a bit confusing for the\nreader. I think it could be corrected by mentioning both the ANOVA and Tukey HSD\nresults in the text, which could quickly become difficult to read, or alternatively by only\ndiscussing the results that were significant according to both statistical tests.\nReply 5-3: We agree that presenting both ANOVA and post-hoc results can be\nconfusing. To ensure clarity and statistical rigor, we have revised the Results and\nDiscussion section to primarily discuss the significance of treatment effects based on\nthe Tukey HSD post-hoc test results where pairwise comparisons are relevant. This\napproach ensures that our textual interpretations align directly with the comparisons\npresented in the figures. We also maintain the reporting of significant main or\ninteractive effects from the ANOVA only when discussing overarching treatment\nimpacts without specific pairwise claims.\nLine comments:\nLine 110 - Suggest starting a new paragraph here (because of the change in subject)\nReply 5-4: Thanks for your suggestion. Done.\nLine 116-118- This example does not follow from the list in the previous sentence, it is\nintroducing an additional idea.\nReply 5-5: Thank you for highlighting this logical inconsistency. We have revised the\nsentences to ensure the example directly illustrates one of the previously listed factors.\n“These inconsistencies likely arise from context-dependent factors such as different\necosystems, host plant identity, and initial AMF community composition. For example,\nelevated temperatures may reduce fungal hyphal growth in arid ecosystems but\nstimulate symbiotic efficiency in cooler regions where warming alleviates microbial\nmetabolic constraints39,40.” Line (112-116).\nLine 121-124- It is unclear how this chain of events links back to the addition of Nr and\nwarming- are these changes in root traits typical following warming or Nr addition?\nReply 5-6: Thank you for this excellent point. We have revised the text to explicitly\nconnect the root trait changes to our experimental treatments.\n“Changes in root traits and AMF communities induced by Nr input and warming are\nlikely to cascade to SOC dynamics, influencing both organic C input and\nstabilization21,41. For example, Nr addition favored roots with high specific root length\n(SRL) and low root diameter or root tissue density (RTD)29. The resulting changes in\nroot trait profiles are often associated with high root turnover or short life span42,\nlimiting their contribution to soil aggregation and subsequent SOC formation43,44.”\n(Line 118-123).\nLine 150- Please specify the diameter and height of the OTCs.\nReply 5-7: Done.\nLine 162 - This statement does not appear to be supported by Table S3, which shows\nno significant relationship between the N treatment and NO3, NH4, or available N.\nReply 5-8: Thank you for pointing this out. We apologize for the typo in the previous\nversion. According to the statistical results (Table S3), Nr input had a significant main\neffect on soil available N (mainly NO -) and also showed a significant interaction with\n3\nwarming and years. Therefore, we further conducted year-specific analysis and found\nthat Nr input significantly increased NO - in each individual year (2018, 2019 and 2021)\n3\n(see Table S2). We have clarified this point in the revised manuscript to avoid confusion.\nLine 163 - \"warming amplified the positive Nr effect on soil available N\" This statement\nappears to be true only in the 3 way interaction with Y (Table S3).\nReply 5-9: Insightful comment. We have revised the sentence as:\n“However, warming amplified the positive Nr effect on soil available N in 2021.” (Line\n162-163).\nLine 165- This again appears unsupported by table S3\nReply 5-10: Thank you for highlighting this. We have revised the sentence based on a\nnew analysis of the available N:P ratio for each year as well as across the three-year\nperiod.\n“Although Nr input did not consistently affect soil available P, it consistently increased\navailable N:P ratio across the three years.” (Line 165-166).\nLine 177 - Figs 1a and 1b are being used as evidence of this statement, but it appears\nthat Fig 1 shows the average across the entire experiment, not the proportions in 2021.\nPlease clarify this in the figure caption for Fig 1 (state more clearly in the title that it is\nshowing an average of all years) and remove Fig. 1 from parenthetical evidence on line\n177.\nReply 5-11: Insightful comment. We have revised the legend of Fig. 1 to clearly state\nthat these values are the average over the 3 years and have also removed Fig. 1 from\nparenthetical evidence.\nLine 185 - Caption should be for Fig. 1f (not 1e). However, in Fig. 1F, Nr does not\nappear to have had any effect on root turnover, contrary to the text.\nReply 5-12: Thanks for pointing out the caption error. It has been corrected. We have\nalso revised the text to accurately reflect the data in the figure: “In contrast, both factors\nhad significant but opposing main effects on root turnover with no significant\ninteraction: Nr input increased root turnover by 54.6%, whereas warming reduced it\nby 30.8% (Fig. 1f; Supplementary Table 4).” (Line 182-185).\nLine 187- It seems that significance is being assigned according to the ANOVA results,\nbut examination of the figure shows no difference in SRL due to Nr according to the\nTukey HSD, as both control and N bars are labeled \"a\" and therefore not significantly\ndifferent. This occurs repeatedly throughout the discussion. I would suggest reporting\nboth that the ANOVA was significant and that the Tukey HSD was not in the text,\notherwise it is confusing as a reader to read something in the text and have contradictory\ninformation in the figures.\nReply 5-13: Sorry for the confusion. We agree that the original wording was confusing\nbecause the significance inferred from the overall ANOVA was not consistently\nsupported by post hoc Tukey’s HSD comparisons shown in the figures. To avoid this\nconfusion, we have revised the text to report only results that are unambiguous and\nconsistent between the statistical analyses and figure annotations.\n“As for fine root functional traits, repeated-measures analysis of variance (ANOVA)\nrevealed no significant interaction between Nr input and warming, but significant\nvariation among years (Supplementary Table 5; Supplementary Figs. 5 and 6).” (Line\n185-187).\nLine 192 - Are these differences in root functional attributes due to a shift in plant\nspecies? Or are the plants are able to alter their root morphology due to the nutrient\nenvironment? Some interpretation of these results by the authors would be helpful.\nReply 5-14: Yes. Our study suggests that these differences in root functional attributes\nare likely due to a shift in plant species.\nLine 199 - \"significantly reduced both parameters\" Flagging again here a discrepancy\nbetween ANOVA results and Tukey HSD results in panel 2h that is not mentioned in\nthe text.\nReply 5-15: Sorry for the confusion. We have revised the text to resolve the discrepancy\nbetween ANOVA results and Tukey HSD results in panel 2h.\n“No significant interaction between Nr input and warming was detected for either\nparameter (Supplementary Table 5).” (Line 197-199).\nLine 200 - Just to repeat that Tukey HSD did not provide support to statement that Nr\nincreased SRL.\nReply 5-16: Thanks. We have now revised the sentence to support our statement.\nLine 202 - Since this is the first time the idea of a \"conservation gradient\" is being\nbrought up, the authors should explain the concept.\nReply 5-17: Sorry for the confusion. We have now explained the concept of\n“conservation gradient”.\n“that is, the classical fast-slow tradeoffs in root economics, where roots with low\nconstruction costs and rapid turnover contrast with more structurally dense,\nconservative root24,26.” (Line 204-206).\nLine 205- Same comment but for \"collaboration gradient.\"\nReply 5-18: Sorry again for the confusion. We have also included an explanation of\n“collaboration gradient”.\n“spanning a continuum from a “do-it-yourself” nutrient acquisition strategy,\ncharacterized by thin, high-SRL roots with low mycorrhizal dependence to an\n“outsourcing” strategy in which plants invest in thicker roots and greater C allocation\nto AMF to acquire nutrients22,23.” (Line 208-211).\nLines 206-210- Further explanation of the significance of the PCA results would be\nhelpful.\nReply 5-19: Good point. We have now made further explanation of the significance of\nthe PCA results.\n“Principal component analysis (PCA) further showed that the first two components\nexplained 72.1% of the variation in mycorrhizal colonization and root traits (Fig. 2j;\nSupplementary Table 10). The first axis (RTD-RNC) captures variation in RTD and N\nconcentration, reflecting differences in root construction costs and nutrient investment\nstrategies. The second axis (RD-AMF%-SRL) describes a gradient from thin roots with\nhigh SRL and low mycorrhizal colonization to thick roots with greater mycorrhizal\ndependence, corresponding to a cooperative root-AMF economic strategy24.” (Line\n211-217).\nLine 221 - I don't believe this statement is supported by the figure. Glomeraceae and\nGigasporaceae in soil both appear unaffected by the N treatment. However there are\nsome interesting differences in the NxW treatment that are not discussed in the text.\nReply 5-20: Thanks for pointing this out. We have revised the text to clarify that Nr\ninput alone did not significantly affect Gigasporaceae in soil. Instead, Nr input showed\na significant main effect on Glomeraceae in soil, whereas a significant interaction\nbetween Nr input and warming was detected only for Gigasporaceae in soil (Fig. 3;\nSupplementary Table 7) (Line 227-233).\nLine 224 - Paraglomeraceae is not changed by the warming treatment contrary to what\nis stated in the text.\nReply 5-21: Sorry for the typo. This has now been corrected.\n“In comparison, warming altered the taxonomic composition of AMF communities in\nroots, with a significant decrease in Glomeraceae across the 3 years, whereas\nParaglomeraceae showed a significant increase only in 2021 (Fig. 3; Supplementary\nTable 7).” (Line 230-233).\nLine 246- I was expecting the AMF results to be given here after the ROOT results.\nReply 5-22: Good point. Done.\nLine 248 - It is unclear what the \"additive\" effect refers to.\nReply 5-23: Sorry for the confusion. We have revised the text to remove the ambiguous\nterm “additive” (also see details in Reply 5-24).\nLine 249 - Again, the significant interaction in the ANOVA did not translate to the figure\nvisually or to the Tukey HSD results.\nReply 5-24: Sorry for the confusion. We have revised the text to better the Turkey HSD\nresults:\n“A significant interaction between Nr input and warming was detected for ROOT-\nderived C, but not for AMF-derived C (Fig. 4a, 4b; Supplementary Table 8). Both Nr\ninput and warming independently reduced AMF-derived C inputs to soil (Fig. 4a).\nRelative to the control, Nr input alone, warming alone and their combination\nsignificantly reduced ROOT-derived C by 38.0%, 40.3% and 41.2%, respectively (Fig.\n4b).” (Line 255-260).\nLine 250 - These results are not supported by the Tukey HSD.\nReply 5-25: Good point. We have revised the sentence to better reflect the statistical\nresults based on the Tukey HSD test.\n“For the MAOC, no significant interaction was detected for either ROOT- or AMF-\nderived MAOC (Fig. 4c, 4d). Instead, Nr input had a significant negative main effect\non both ROOT- and AMF-derived MAOC, while warming exerted a significant negative\nmain effect only on AMF-derived MAOC (Fig. 4c, 4d). However, a significant\ninteraction between Nr input and AMF presence was detected for total soil MAOC\n(Supplementary Fig. 12b) and Ca-associated organic C (Ca-OC) (Fig. 5a;\nSupplementary Table 9).” (Line 261-266).\nLine 253- The use of \"and/or\" is unclear here, since the three treatments are no roots or\nAMF, AMF only, or roots + AMF.\nReply 5-26: Sorry for the confusion. We have revised the sentence to clearly reflect the\nthree treatment levels:\n“However, a significant interaction between Nr input and AMF presence was detected\nfor total soil MAOC (Supplementary Fig. 12b) and Ca-associated organic C (Ca-OC)\n(Fig. 5a; Supplementary Table 9).” (Line 264-266).\nLine 258 - The figure does not indicate a significant interaction.\nReply 5-27: Thanks for pointing this out. We have revised the word “interaction” to\n“combination” to more accurately reflect the results shown in the figure.\nLine 260 - I don't believe this regression analysis is described in the Statistical methods.\nReply 5-28: You are exactly right. We have now added a description of this regression\nanalysis to the Statistical methods section.\nLine 281- How does this result compare with prior studies?\nReply 5-29: Good question. Our previous study (Qiu et al., 2021), as well as the work\nby Erktan et al., (2016) and Ding et al., (2021) reported that plant tends to construct\nthinner, longer roots, which enhance rhizosphere priming and stimulate SOC\ndecomposition.\nReferences:\nDing, W., Cong, W. & Lambers, H. Plant phosphorus-acquisition and -use strategies\na strategies\naffect soil carbon cycling. Trends Ecol. Evol. 36, 899–906 (2021).\nErktan, A. et al. Increase in soil aggregate stability along a Mediterranean successional\ngradient in severely eroded gully bed ecosystems: combined effects of soil, root traits\nand plant community characteristics. Plant Soil. 398, 121–137 (2016).\nQiu, Y. et al. Warming and elevated ozone induce tradeoffs between fine roots and\nmycorrhizal fungi and stimulate organic carbon decomposition. Sci. Adv. 7,\neabe9256 (2021).\nLine 288 - I very much enjoyed reading the end of the discussion and appreciate the\nsynthesis of ideas presented.\nReply 5-30: Thank you very much for your positive comment and encouragement.\nLine 299 - Please explain what is meant by the \"fast and slow return.\"\nReply 5-31: Sorry for the confusion. Fine-root traits are thought to align along a root\neconomics spectrum, ranging from a fast return strategy characterized by thin,\nmetabolically active roots with rapid resource acquisition to a slow return strategy\ncharacterized by thicker, denser roots with longer life spans and sustained returns on\nconstruction costs (Reich, 2014; Bergman et al., 2020).\nReferences:\nReich, P. B. The world-wide `fast-slow’ plant economics spectrum: A traits manifesto. J.\nEcol. 102, 275–301 (2014).\nBergmann, J. et al. The fungal collaboration gradient dominates the root economics\nspace in plants. Sci. Adv. 6, eaba375 (2020).\nLine 302- Suggest specifying in this sentence that \"This indicates that plants\nexperiencing Nr additions may optimize their C allocation belowground for resource\nacquisition...\"\nReply 5-32: Good suggestion. Done (Line 316-318).\nLine 304- \"Finally, a shift...\" Please specify if the shift was due to Nr or warming or\nboth.\nReply 5-33: Sorry for the confusion. We have revised the sentence to: “Finally, Nr\ninput and warming induced shift” to explicitly clarify the shift was driven by both\nfactors.\nLine 328 - Should \"production\" be \"protection\"?\nReply 5-34: Good point. We have revised “production” to “protection” as suggested.\nLine 344- \"Taking\" should be \"Taken\".\nReply 5-35: Corrected.\nFig. 2- In the PCA panel, should \"D\" be \"RD\"?\nReply 5-36: Sorry for the typo. Corrected.\nFig. 6 - Very helpful figure! Suggest adding a panel for the Nr x Warming treatment.\nReply 5-37: Thanks. Done.\nMethods - This study necessarily includes many different methods. It would be helpful\nto have a brief introductory paragraph in the methods providing an overview of the\nmethods used to test the paper's hypotheses.\nReply 5-38: Good suggestion. We have now added a brief introductory paragraph in\nthe Methods section that provides an overview of the major methodological approaches\nused to test our hypotheses (See Line 426-428; 498-501; 534-535; 547-548; 559-561;\n577-578).\nLine 443-446- Suggest briefly including here the information in the supplement\ndescribing the ratio and why it can be used as indicative of AMF.\nReply 5-39: Good point. We have now explained why the ratio can be used as indicative\nof AMF (see Supplementary Method).\n“NLFA 16:1ω5 is generally regarded as a more sensitive indicator of AMF mycelial\nbiomass than PLFA 16:1ω5 because the ratio between NLFA and PLFA 16:1ω5 is\ntypically high in AM fungi (ranging from 1 to 200), but consistently low in bacteria (<1)\n(Olsson, 1999; Olsson and Johansen, 2000). In the present study, the NLFA/PLFA\n16:1ω5 ratio ranged from 2 to 8 (Supplementary Fig. 7g), supporting the use of PLFA\n16:1ω5 as reliable proxy for AMF biomass. Notably, the effect of Nr input on NLFA and\nPLFA 16:1ω5 were highly consistent (Supplementary Fig. 7f, h), further validating the\nrobustness of our AMF biomass estimates.”\nLine 565 - This appears to be an incomplete sentence\nReply 5-40: Thanks. We have revised the sentence.\n“It is worth noting that any additional C from roots and AMF may affect the final 13C\nvalue of the retrieved soil-residue mixture from field.” (Line 589-591).\nReviewer #6 (Remarks to the Author):\nIn the paper “Root traits and mycorrhizal fungal community mediate reactive N and\nwarming impacts on soil organic carbon” by Qiu and co-authors, the authors present\nfinding from a multi-year N x warming experiment in a semi-arid grassland. Their goal\nwas to isolate the mediating effects of belowground plant and fungal traits. They found\nmany changes in AMF community composition, root morphological traits, root\nturnover, and—importantly—root-derived and fungal-derived C formation. The paper\nis interesting, but my primary goal here, rather than giving an additional thorough\nreview—is to follow up on reviewer #3’s concerns about root-derived C input\ncalculations. I also include a couple minor comments that came up as I read through the\nwhole paper.\nOverall, I am satisfied with the authors’ response to R3’s concerns about root-derived\ninput calculations and about the potential role of DOC lateral flow. Results from this\ntype of multi-mesh size ingrowth core approach have been published by numerous\ninvestigators. It is not without flaw, but it is among the best methods available for\ncharacterizing belowground inputs in the field. My only concern is a minor one. Even\nafter reading the paper and response, I am still confused about whether ROOT-derived\nincludes C derived via the AMF hyphae. I.e. Is it everything (total root and root-\nassociated fungus) or total inputs from the root ingrowth core minus inputs from the\nhyphal ingrowth core? I ask because, as the authors indicate, it is impossible to fully\nisolate the AMF vs root-derived C as there is some AMF biomass in the root that would\nnot be captured by the hyphal ingrowth cores. This is not a big deal for their take home\npoints, but it would help the reader to discuss this very briefly.\nReply 6-1: Thank you for your positive comments and raising this important point for\nclarification. As you correctly note, complete separation of ROOT- and AMF-derived\nC is not possible due to intraradical fungal structures.\nIn this study, ROOT-derived C represents the total C inputs associated with roots,\nincluding both root tissue-derived C and C from root-associated (intraradical and\nextraradical) AMF. To clarify this distinction for readers, we have revised the Method\nand Supplementary Figure 2 (see below) to more explicitly illustrate how ROOT- and\nAMF-derived C inputs were defined and calculated.\n“To isolate the effects of roots and their associated AMF on SOC formation and stability,\nwe established a C plant-C soil system using root- and hyphae-ingrowth cores (Keller\n3 4\net al., 2021). Three types of cores (Supplementary Figure 2) were installed in each plot\n(Xu et al., 2022): (i) no-hyphae control (0.45 μm mesh), excluding both roots and AMF\nhyphae; (ii) hyphae-ingrowth cores (25 μm mesh), permitting only AMF hyphae\npenetration; and (iii) ROOT-ingrowth cores (1.8 mm mesh), allowing both roots and\nAMF hyphae to penetrate. The net effect of the root and AMF pathway on SOC or\nMAOC was calculated as (Supplementary Figure 2):\nAMF-derived effect = (hyphae-ingrowth cores, B) − (no-hyphae control, A)\nROOT-derived effect = (ROOT-ingrowth cores, C) − (no-hyphae control, A)”\nA couple of minor points:\nI would avoid the impression that MAOC is a simple indicator of SOC stability. Yes,\nMAOC has a longer residence time, but there is a lot we still don’t know about whether\nrecently formed MAOC is “stable”.\nReply 6-2: Good point. We agree that MAOC should not be interpreted as a simple or\nabsolute indicator of SOC stability, particularly given growing evidence that recently\nformed MAOC may remain dynamic and potentially labile (Kleber et al., 2021; Díaz-\nMartínez et al., 2024). We have therefore revised the manuscript to avoid language\nimplying that MAOC alone equates to SOC stability. Instead, in this study, we used\nMAOC alongside microbial necromass C as two complementary proxies to infer\nchanges in SOC stability.\nReferences:\nKleber, M. et al. Dynamic interactions at the mineral-organic matter interface. Nat. Rev.\nEarth Environ. 2, 402–421 (2021).\nDíaz-Martínez, P. et al. Vulnerability of mineral-associated soil organic carbon to\nclimate across global drylands. Nat. Clim. Change 14, 976–982 (2024).\nI recommend mentioning any essential methodological details in the last paragraph of\nthe intro or in the results. I’m thinking mostly of plot size here, which is important for\ninterpreting plant community changes.\nReply 6-3: Thank you for this valuable suggestion. We have added this essential detail\nabout plot size, which will help readers better interpret the scale and ecological context\nof our study (see Line 148 and 150)."}]

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