Positive rhizosphere priming accelerates carbon release from permafrost soils

Domain: earth_science
Published: 2025-04-15
Editorial outcome: minor_revision

Abstract
Thawing permafrost soils are predicted to release substantial amounts of carbon by 2100. In addition to this, warming-induced active-layer deepening and increased rooting depth may result in further carbon losses from previously-frozen soil by stimulating microbial communities through fresh carbon inputs inducing positive rhizosphere priming. While models based on temperate data predict significant permafrost carbon loss through rhizosphere priming, data from permafrost soils are lacking. Here, we provide direct evidence of live plant-induced positive rhizosphere priming in permafrost and active-layer soils across diverse soil types from Arctic and Subarctic Canada. By 13CO2 labelling plants in a controlled environment, we show that root activity increases carbon loss from previously frozen soils by 31%. This rhizosphere priming effect persists longer in permafrost than in active-layer soils, suggesting greater vulnerability of permafrost carbon. These findings underscore the urgency of incorporating plant–soil–microbe interactions into models predicting greenhouse gas emissions from thawing permafrost.

Introduction
Northern permafrost regions are one of the largest and most vulnerable stores of soil organic carbon (SOC) globally1,2. These frozen carbon (C) stores are threatened by rising global temperatures leading to permafrost thaw, which may release 10s of billions of tonnes of C by 21003,4. Due to widespread increases in the depth of seasonal thaw as a result of warming, near-surface permafrost is thawing in many polar regions5. When such active-layer deepening occurs, a previously unoccupied soil volume becomes available for plant roots that invade newly-thawed permafrost6, with experimental evidence showing that plants access available nutrients at the thaw front7,8. Furthermore, it has been shown that roots found in current permafrost soils grew downwards through newly-thawed soil during the early Holocene warm interval in the western Arctic, prior to refreezing within an aggrading permafrost in the mid-late Holocene9. Importantly, modern permafrost thaw coupled with greater plant productivity10 and increased rooting depths may increase rates of C inputs to soil in permafrost regions. Based on observations from temperate regions, there are predictions that greater C inputs could stimulate decomposition of previously-frozen soil organic matter (SOM) by a mechanism known as the rhizosphere priming effect (RPE), but direct evidence for this effect remains limited with few empirical data on live plant-induced RPEs in permafrost environments.

The RPE represents a change in SOM decomposition induced by plant roots compared to soil without roots, and is caused by plant root activity supplying soil microorganisms with energy-rich compounds such as root exudates and litter11. The RPE can be positive (accelerating SOM decomposition) or negative (when root activity suppresses SOM decomposition).

Positive RPEs may occur when microorganisms utilize fresh C as an energy source to produce extracellular enzymes which facilitate the decomposition of complex organic molecules12 to access and metabolise C and nitrogen (N). One example of this is ‘N-mining’, where extracellular enzymes are deployed to release nutrients from complex organic matter, and is one of the proposed mechanisms for arctic RPEs13,14 corroborated by the absence of positive RPEs in plots dominated by N-fixing Alder15. This implies that RPEs may be controlled by microbial nutrient demand16, however evidence from incubation experiments suggests that microbial N-mining is unlikely to be the sole mechanism driving positive priming17,18. Alternatively, RPEs may be driven by SOM quality and microbial C and energy availability12. Negative RPEs may occur if microbes shift to preferentially utilising fresh C inputs rather than SOM or because increased competition for nutrients between plant roots and microbes decreases microbial growth and metabolism, thereby depressing SOM decomposition11. These proposed mechanisms for RPEs11 remain largely untested in permafrost soils.

Radiocarbon evidence from field studies suggests that arctic soils are vulnerable to RPEs15,19. Furthermore, recent modelling work indicates that RPEs may amplify overall soil respiration in permafrost-affected ecosystems by ~12% and could increase absolute soil C loss from northern permafrost regions by ~40 Pg soil C by 210020. However, the model could not directly parameterise priming effects induced by live plants in Arctic or Subarctic soils due to lack of data from these regions. Research into the priming of permafrost soils has been carried out using incubation studies in soil-only systems with C supplied not through plants but as labile substrate additions21–26. However, this method may miss the chemical, temporal and spatial complexity of live plant-delivered root exudates27, and lacks a key sink for nutrients, namely plant biomass. Several proposed mechanisms driving both positive and negative RPEs depend on competition for nutrients either between microbes or between plants and microbes16,28 which likely differs fundamentally in plant–soil systems compared with soil-only systems. Therefore, there is a critical knowledge gap in understanding the genuine role that plant-derived C plays in priming decomposition of SOM and increasing C release from permafrost soils as thaw depths (and hence rooting depths) increase.

We hypothesise that i) fresh plant-C inputs into previously frozen soils through root exudation will stimulate the microbial community, causing greater mineralisation of old C from soils with roots compared to root-free controls; and ii) positive RPEs will be driven by microbial nutrient demand rather than being due directly to the energy provided by fresh C inputs.

We quantified RPEs induced by live plants growing in a range of permafrost soils, including both active layer and permafrost horizons from contrasting soils representative of large areas of northern permafrost regions. To achieve this, we developed a bespoke 13C-labelling plant-growth chamber29 capable of i) maintaining constant 13CO2 atmospheric enrichment of 500‰ across fixed day–night cycles for 370 days, equivalent to about five Arctic growing seasons; ii) controlling soil temperatures substantially below air temperatures, as experienced in Arctic summers; and iii) quantifying soil CO2 efflux and its 13C signature from 216 experimental units. The use of 13CO2 and soils with and without roots allow partitioning of C effluxes from soil into i) plant-derived C and ii) RPE-induced release of soil C. Our experimental design also investigated the relative importance of live roots versus root litter in controlling priming effects, as well as the role of nutrient availability in controlling the direction and/or magnitude of priming effects.

Here we show that live plant roots significantly enhance SOM decomposition in both permafrost and active layer soils, leading to net C loss through rhizosphere priming (positive RPE). Across a range of Arctic and Subarctic soils, the presence of roots increased SOM-derived CO2 flux by an average factor of 1.31 over 370 days. In active layer soils, priming effects were strongest during the first 185 days before declining. In contrast, permafrost soils exhibited sustained and increasing priming effects through time. The persistence of priming in permafrost suggests that positive RPEs in thawing permafrost soils may increase C losses, amplifying the effect of permafrost thaw on climate change.

Results
Positive RPEs in active layer and permafrost soils
Soil types from dominant suborders of permafrost-affected soils (Gelisols, i.e., Orthels, Turbels) were collected along a transect across continuous and extensive discontinuous permafrost in northwest Canada (site descriptions in Supplementary Information). The soils developed on a variety of major Quaternary superficial deposits, including silt- and clay-rich till, sandy alluvium, silt-rich yedoma and organic-rich thermokarst-lake-basin deposits. They cover the main types of permafrost terrain (glaciated vs non-glaciated) and growth (epigenetic vs syngenetic) in the Arctic and Subarctic.

The collected permafrost and active layer soils were used to investigate rhizosphere priming effects in permafrost region soils within a temperature controlled, 13C-labelling plant-growth chamber29. By measuring CO2 fluxes and 13C signatures from the headspace of both rooted and root-free compartments of custom-designed mesocosms (see Methods for details), we were able to partition soil C effluxes into plant-derived CO2 and SOM-derived CO2 thereby quantifying the RPE-induced release of soil C.

Across the wider range of permafrost and active layer soils tested here (Supplementary Fig. 1) the presence of roots caused greater fluxes of SOM-derived CO2 and RPE ratios > 1, resulting in net C release from both active layer and permafrost soils (Fig. 1). Our data from all soils and over the full duration of the experiment since plant germination (370 days) show that SOM-derived CO2 flux from soils with roots was on average higher than from root-free soil by a factor of 1.31 (1.22–1.40 95% CI; RPE ratio; Fig. 1a). No discernible patterns were observed in the RPE data between soil types (Fig. 2).

Severing ingrown roots—thereby halting the supply of fresh root exudates—results in a drop of SOM-derived CO2 flux within one month (Supplementary Fig. 3b, c), suggesting rhizosphere priming was largely driven by fresh root exudates, rather than root litter. The drop in SOM-derived CO2 flux induced by root severing likely occurred sooner than one month, as root exudates are turned over and respired within days30 and therefore likely to have a short-lived impact on SOM-derived CO2 flux. However, with our monthly flux measurements we are unable to quantify precisely how quickly positive priming effects were lost. The observed drop in SOM-derived CO2 flux following the severing of live roots corroborates evidence from work which found that synthetic root exudates can induce soil C loss through soil priming and mobilisation of soil C24,27,31,32. This experimental observation is significant for modelling the magnitude and spatial distribution of RPEs in permafrost regions, with many areas seeing significant increases in above-ground biomass (arctic “greening”) and therefore fresh soil C inputs, along with areas which are browning (decline in biomass) and burning in spatially heterogeneous ways33 with likely impacts on belowground delivery of fresh C in both space and time.

In active layer soils, positive RPEs occurred in the first 185 days (Fig. 1c, d, p<0.05 on days 56 & 173), in contrast to results from incubation studies with labile substrate additions which find no priming of active layer soils22,23. These differing experimental results may indicate that active layer soils are vulnerable to priming by live root activity but not by the addition of labile substrates. The RPE ratio in active layer soils was 1.39 (1.21–1.57 95% CI; Fig. 1c) in days 0–185 and 1.03 (0.89–1.17 95% CI; Fig. 1c) between days 186 and 370. This change in the magnitude of RPEs over time suggests that there may only be a small pool of C in active layer soils that is vulnerable to priming. Alternatively, the result could be linked to reduced plant growth later in the experiment; less frequent clipping of above-ground biomass was necessary in the later parts of the experiment. However, we consider the plant growth explanation to be less likely because the reduction in positive priming beyond day 186 was not observed in permafrost soils, which had the same above-ground plant growth trends. In addition, headspace CO2 concentrations did not decline substantially over time, indicating that root activity remained high (Supplementary Fig 3a). In contrast, in permafrost soils positive rhizosphere priming occurs throughout the experiment (Fig. 1d, e, p<0.05 for most but not all measured days), with an RPE ratio of 1.39 (1.29–1.50 95% CI; Fig. 1e), and RPE ratios greater than 1.5 observed from day 300. After day 250, permafrost soils had a significantly higher RPE ratio than active layer soils (p=0.0035, F=5.21, df=3). The maintenance and potential increase in priming in permafrost soils later in the experiment, in contrast to the decline in priming over time in active layer soils, suggest that permafrost soils may be more vulnerable than active layer soils to positive RPEs through time. Given that the majority of soil C in northern permafrost regions is stored below depths of 30 cm, the potential for sustained positive RPEs in permafrost soils and increased mineralisation of this C could amplify the positive feedback between permafrost CO2 emissions and global warming4,34.

Factors controlling the magnitude of positive RPEs
The greater and more persistent positive priming effects observed in permafrost soils may arise if the permafrost microbial community in older, deeper soil layers, with more degraded SOM, are more limited by labile C and energy availability12 than active layer soils. Consistent with this explanation, rates of CO2 release per unit C were lower in permafrost than active layer soils (p=0.029, F=5.395, df=1, Fig. 2d, e). Stronger microbial C limitation has been linked to greater priming effects following 13C-labelled substrate additions in permafrost soils26. The RPE ratio was significantly negatively related to cumulative SOM-derived CO2 flux for all soils (p=0.0015, F=12.37, df=29). This suggests that the greatest priming effects occurred in soils with low bioavailable SOM, consistent with stronger microbial C limitation being related to increased priming intensity.

The difference in RPEs over time between active layer and permafrost soils may alternatively or additionally be driven by the mobilisation of physico–chemically protected organic matter in mineral permafrost soils. Root exudates, specifically those containing organic acids, have been found to liberate organic matter from mineral association thereby making it microbially available32. As a greater proportion of SOC has been shown to be minerally-associated in deeper mineral permafrost soils than in organic-rich active layer soils, this effect could be more pronounced in permafrost soils and may explain the patterns in RPEs observed here. However, no relationships were found between RPEs and soil primary texture in this experiment (Supplementary Fig. 1d, e). Further experiments are needed to investigate these mechanisms fully and to elucidate the extent to which positive RPEs in permafrost are driven by microbial C limitation or direct action of plant derived organic acids.

The soils used here covered a wide range of %C, %N and C:N ratios (Supplementary Fig. 1a), allowing further investigation of factors that have been implicated in driving the magnitude of priming effects12,24,35. Positive rhizosphere priming was observed across soils with differing C contents, N contents and C:N ratios. However, the RPE ratio exhibited a significant negative power-law relationship with soil C and N content across all soils (C; p=0.0016, F=12.18, df=29, N; p=0.0047, F=9.37, df=29) and in permafrost (C; p=0.027, F=2.76, df=16, N; p=0.021, F=2.90, df=16) but not active layer (C; p=0.45, F=1.10, df=11, N; p=0.35, F=1.27, df=11) soils (Fig. 2a, b). Across the full experimental duration, at a given C or N content, RPEs tended to be greater in permafrost horizons, but this effect was only statistically significant for N (C; F=3.64, p=0.067, N; F=4.42, p=0.044). This indicates that organic matter in organic-rich active layer and permafrost soils may be less vulnerable to priming, which could be driven by lower microbial C or nutrient limitation reducing the intensity of soil priming26 in these soils.

The RPE ratio was significantly positively related to soil C:N ratio in active layer soils (R2=0.38, p=0.024) but shows no relationship in permafrost soil (R2=0.04, p=0.42) or across all soils (R2=0.03, p=0.95). Across the full experimental duration, at a given C:N ratio, RPEs were not significantly different between active layer and permafrost soils (Fig. 2c; F=2.93, p=0.10). It has been suggested that if RPEs are driven by microbial C limitation then only soils with a C:N ratio below 20 should be susceptible to RPEs20,26 due to the stoichiometric constraints of microbial growth. However, here we observe positive RPEs in soils with C:N ratios up to 37.6 (Fig. 2c). Positive priming in Arctic soils with high C:N ratios has previously been observed36, although this was in high-C-content peat soils, which was not the case here. The observed positive relationship between RPEs and soil C:N ratio in active layer soils here suggests that N availability plays a role in driving RPEs in active layer soils; soils with lower relative N exhibited the strongest positive priming effects, indicating that when C is available, but N is limiting, active layer soils are most vulnerable to C loss through positive RPE. Priming of active layer soils with high C:N ratios may therefore be caused by microbial and plant competition for nutrient acquisition in soils with high C and low N contents, i.e. microbial and plant N limitation as opposed to microbial C limitation. Overall, there appear to be evidence for both microbial C and microbial or plant nutrient limitation playing key roles in controlling the magnitude of priming effects, with a potentially greater role for C limitation in some permafrost soils, especially turbels. Detailed experiments are needed to investigate these mechanisms fully.

Nutrient additions temporarily reduce RPEs
To further test the potential for different mechanisms driving RPEs in active layer versus permafrost layers, we supplied active layer and permafrost soils with additional nutrients (equivalent to ten times microbial biomass N) once, at the beginning of the experiment in a slow release form to maintain nutrient abundance in these soils for as long as possible through the duration of the experiment (we identified small amounts of unused fertiliser in the soils at the end of the experiment). These high levels of nutrient addition were added to enable the reduction of microbial nutrient limitation in the presence of strong plant growth and therefore plant nutrient uptake.

Overall, the magnitude of RPEs in permafrost soils was reduced in the presence of additional nutrients compared to soils without nutrient addition (p=0.0037, F=8.53, Figs. 1 and 3). This was particularly prevalent early in the experimental period. There was no significant positive priming in soils with added nutrients in the initial 100 days as soils with roots had similar SOM-derived CO2 fluxes to soils without roots. The absence of a significant RPE ratio over this period (RPE ratio=1.06, 0.76–1.37 95% CI; Fig. 3a) indicates that high nutrient levels may reduce RPEs initially. This agrees with previous work identifying lower priming intensity in permafrost soils with greater N availability or following N addition in experiments using soil incubation with 13C-labelled substrates under thaw-induced increased N availability and N addition37. Furthermore, relationships between RPE ratio and soil C content, N content and C:N ratio (Fig. 2) were lost in the presence of additional nutrients (Fig. 4). RPE ratios in soils with additional nutrients was not related to soil C content, N content or C:N ratio (prior to nutrient addition) in both active layer (C; R2<0.001, p=0.97, N; R2=0.001, p=0.92, C:N; R2=0.002, p=0.89) and permafrost (C; R2=0.14, p=0.20, N; R2=0.15, p=0.19, C:N; R2=0.12, p=0.24) soils (Fig. 4a–c). At a given C content, N content or C:N ratio, in the presence of additional nutrients RPEs were no longer significantly different between active layer and permafrost soils (C; F=1.56, p=0.22, N; F=0.74, p=0.40, C:N; F=0.38). This suggests that some of the key differences between permafrost and active layer soils in terms of the magnitude of their RPEs was related to their nutrient status.

The observed reduction in RPEs in soils with nutrient addition is likely driven by an alleviation of microbial N-limitation leading to less microbial N-mining in these soils and resulting in the reduction or absence of a positive priming effect. Alternatively, the observed reduction in RPEs in soils with nutrient addition may also be driven by alleviations of plant N-limitation38, which may have reduced resource allocation to root strategies for accessing low availability nutrient pools including exudation or organic acids and enzymes. Overall, in the permafrost and active layer soils with nutrient addition, the nutrient demand may be matched by the supply of excess nutrients, at least initially, reducing the need for N-mining and, hence reducing positive priming effects.

Over time, it is likely that the available nutrient pool diminished, and plant nutrient demands may have started to exceed available nutrient supply in the soils. In line with such a shift; a return to positive RPEs was observed in days 101–370 (RPE ratio=1.33, 1.25–1.41 95% CI; Fig. 3a). This temporal effect was consistent across both active layer (RPE 1.30, 1.11–1.48 95% CI; Fig. 3c) and permafrost (RPE 1.38, 1.27–1.48 95% CI; Fig. 3e) soils, returning to a positive RPE after 185 days. It is also possible that changing soil nutrient status altered the composition and function of the microbial community39, which may have altered the magnitude of priming effects over time. The overall reduction in the magnitude of RPEs with additional nutrients in both active layer and permafrost soils suggests that plant and microbial nutrient demand contributed to positive priming effects in both soil layers. However, based on the changes in RPEs over time in our experiment, it is likely that high levels of nutrient release associated with permafrost thaw7,8 may merely delay the onset of positive RPEs rather than mitigate them entirely. Future studies that monitor changes in microbial community composition and function in response to changes in nutrient availability would be a valuable addition and could help to understand the relative role of microbial versus plant nutrient demand in shaping RPEs, as well as the relative roles of microbial C versus nutrient limitation.

Implications for predicting the magnitude permafrost feedback
Our results collectively suggest that RPEs in permafrost and active layer soils are governed by both the limited availability of high energy substrates to support microbial activity and the restricted availability of nutrients to sustain plant growth. This dual limitation highlights a complex interplay where microbial metabolism is hindered by insufficient readily decomposable organic matter, which can be alleviated by fresh C inputs from plant roots, thus activating microbes which decompose SOM. In addition, the plants also compete for nutrients, critical to sustaining their productivity, resulting in the breakdown of complex organic matter. The positive RPEs that we have observed thus represent interactions between plant nutrient limitation and microbial C and nutrient limitation. Further study is required to identify the relative importance of the different mechanisms in different permafrost soil types. Studies that measure the responses of microbial growth and C use efficiency to nutrient and C availability in permafrost may be critical in this area38, with the understanding generated in combination with priming experiments likely to be crucial for predicting rates of permafrost C loss under changing climatic conditions.

In summary, we show that plant-root activity could substantially increase (by a factor of 1.31 in our study) the release of previously-frozen C following permafrost thaw. While increased carbon losses also need to be placed in the context of any changes in the rate of new SOM formation, this direct quantification of RPEs critically demonstrates that RPEs are induced by fresh C supplied by live plants. Furthermore, we have demonstrated that positive RPEs occur across contrasting soils that developed on different Quaternary superficial deposits, in different types of permafrost terrain (glaciated vs non-glaciated) and which have formed through distinct processes. Thus, RPEs are likely to increase permafrost C losses overall, and should be included in model projections of the permafrost C feedbacks and their implications for the global C budget.

Methods
Soilsampling
The field area selected was a transect in northwest Canada from Tuktoyaktuk on the Arctic Ocean coast through the East Channel region of the Mackenzie Delta near Inuvik, NT, across Peel Plateau, NT, to the Klondike Goldfields of interior Yukon Territory. The transect crosses from continuous permafrost near Tuktoyaktuk and in the Mackenzie Delta and Peel Plateau to extensive discontinuous permafrost in the Klondike. It also crosses from tundra in the Tuktoyaktuk Coastlands and Peel Plateau to boreal forest in the Mackenzie Delta and Klondike. The Quaternary geology and environmental context of the permafrost deposits are well known in this region40–42. Large vertical sections through permafrost deposits are well exposed along the coast, in lake margins, along river bluffs, in headwalls of retrogressive thaw slumps and in valley-floor gold mines, facilitating stratigraphic understanding of the soil profiles43. Samples were collected in August 2019 and August–September 2020 (Supplementary Table 2 & Supplementary Figs. 4–60).

Four types of permafrost soil profiles were sampled: (1) non-lake orthels, (2) drained thermokarst-lake sediments, (3) turbels, and (4) yedoma deposits (Supplementary Tables 1 and 2). The rationale was threefold: first, to compare examples from the main types of permafrost soils (Gelisols) i.e., Turbel, Orthel; second, to compare soils developed on epigenetic permafrost represented by stable till surfaces with syngenetic permafrost represented by aggraded sedimentary deposits (yedoma silt in the Klondike and alluvial silt and sand in the Mackenzie Delta); and third, to compare soils developed in drained thermokarst-lake basins (an integral part of lowland, ice-rich permafrost terrain, which often preferentially accumulate soil organic carbon) with non-lake profiles.

The occurrence of dead roots within epigenetic permafrost is widespread along the western Arctic coast, Canada, and reflects plant growth in a deeper-than-present active layer during the early Holocene9. It is therefore clear that roots grew downward through newly thawed soil in a deepening active layer during the last major episode of global warming during the last glacial-to-interglacial transition prior to refreezing within aggrading permafrost in the mid to late Holocene.

All collected soil samples where frozen upon return to the field station and shipped frozen to the University of Exeter where they remained in frozen storage until processed for the beginning of the experiment.

Soilsampleanalyses
Soil C and N content was quantified by running a 15 mg (+/− 0.5 mg) of each homogenised and ground soil sample on a Flash 2000 Elemental Analyser (Thermo Scientific). Ethylenediaminetetraacetic acid (EDTA) was used as standards at the start of the CN run and as ‘check standards’ every ten samples. The EDTA standard is 9.59% N (+/− 3% accepted) and 41.1% C (+/− 1% accepted).

Soil primary texture analysis on all samples was measured on a Bettersizer S3+ (Bettersize Instruments Ltd., Dandong, China) particle analyser. Organic material was removed from 1g of soil by digestion in 10 ml 30% hydrogen peroxide at 100 °C for 2–3 h until the supernatant was clear. Samples were then suspended in 1% sodium hexametaphosphate to aid dispersion and sonicated at low energy immediately prior to analysis.

Mesocosmdesign
The mesocosms for this experiment were constructed to enable plant growth in an inert substrate in a central compartment with root access via a 2 mm nylon mesh to three soil-filled side compartments. Mesocosms were built using 5 mm thick grey PCV sheet machine cut to size and glued together using UNI-100 PVC cement (Griffon, Bolton Adhesives, Rotterdam, NL). All joints were sealed with Aquamate, a solvent free aquarium sealant (Everbuild, Sika, UK). The whole mesocosm measured 30 cm L × 20 cm W × 20 cm H. The central compartment was 10 cm L × 2 cm W × 20 cm H. Each of the four side compartments was 10 cm L × 10 cm W × 20 cm H and filled to 10 cm with soil, creating a ~1 L headspace (10 × 10 × 10 cm).

The side compartments were sealed with lids cut from 5 mm thick PVC sheet spanning two side compartments within sets of 10 × 10 cm to create a two-sided surface for the lid to seal onto the mesocosm base. An airtight seal was achieved using non-setting putty (Plumbers Mait, EVO-STIK, UK) between the mesocosm base and lid. Two holes in the mesocosm lid allowed the entry of a 6 mm O.D. inlet tube and a 9 mm I.D. outlet tube for each side compartment.

Experimentaldesign
A total of 54 mesocosms were used in this experiment. Thirty-one mesocosms had a temporal root exclusion mesh regime and included the broadest possible range of soil types, C and N content and texture. The remaining 23 mesocosms had additional nutrients added to the soils (Supplementary Fig. 61). Whilst we did not have the space to incorporate all 31 soils into the nutrient addition part of the experiment, the 23 soils selected covered the full range of soil types, C and N content and soil textures. The statistical effects of the uneven n number were checked and found to have no significant effect.

In the mesh regime mesocosms one side compartment had a 1 µm root exclusion nylon mesh (Normesh Ltd, Lancaster, UK) inserted from the beginning of the experiment (12th June 2023) and remained throughout, creating a root-free control. To investigate the relative effects of C inputs from live roots, and associated plant nutrient uptake, versus C and nutrients input through fresh root litter, in two of the compartments the roots were severed at different times points.

To achieve this a second root-exclusion mesh was inserted into the side compartment diagonally opposite the root-free control side compartment on 14th November 2023, and a further root-exclusion mesh was inserted into the side compartment next to the root-free control side compartment on 25th March 2024. Prior to insertion of the mesh, the roots grown into the side compartment soil were severed with a knife, creating root necromass/litter. Once inserted all root-exclusion meshes remained in place until the end of the experiment 11th July 2024.

In the mesocosms with additional nutrients, one side compartment had a 1 µm root exclusion nylon mesh (Normesh Ltd, Lancaster, UK) inserted from the beginning of the experiment (12th June 2023) and remained throughout, creating a root-free control. The remaining three side compartments had continuous root access throughout the duration of the experiment. Additional nutrients were added to these soils once at the beginning of the experiment (12th June 2023) in the form of slow release Velvit Nutrilong V180 controlled release compound fertiliser (29% N, 4% P, 8% K, 1.7% MgO, C:N=0.51), chosen for its 6-month release period and low C content. Additionally, permafrost thaw releases a range of nutrients44,45 which we have tried to capture with this compound input. The amount of fertiliser added to each side compartment was tailored to the soil within each mesocosm. Of the three rooted side compartments, one had no additional nutrients, one had “low” additional nutrients equivalent to 2× microbial biomass N, and one had “high” additional nutrients equivalent to 10× microbial biomass N. By making the nutrient addition proportional to microbial biomass we aimed to alleviate microbial nutrient demand. The root-free control side compartment was given the same nutrient treatment as the high additional nutrient side compartment (Supplementary Fig. 61). Despite previous studies not observing strong direct effects of nutrient additions on respiration in root-free soils46, in our experiment, the “high” additional nutrient soils showed higher fluxes, therefore we limited our analysis to the “high” nutrients, with and without roots.

Plantgrowthandmaintenance
Agrostis capillaris was grown in the mesocosm central compartment to provide the delivery of fresh C inputs to previously frozen soils in the surrounding compartments. Although A. capillaris does not represent the varied vegetation types at field sampling sites (Supplementary Figs. 4–60) it was used as it combines a distribution that includes high northern latitudes with high germination rates from seed and an ability to establish and grow well in growth chamber studies. This was deemed the best combination for geographical relevance and experimental application within the physical and temporal constraints of a growth chamber experiment.

Agrostis capillaris seeds were sterilised using a 30% hydrogen peroxide solution. All seeds were suspended in a 30% hydrogen peroxide solution for 10 min and then rinsed thoroughly with mili-Q water. This was repeated three times before drying the seeds for 48 h at room temperature.

The central compartment of each mesocosm was filled with Field and Fairway Ceramics (Profile, Buffalo Grove, Illinois) as this inert substrate demonstrated good water retention, resistance to compaction and would encourage root growth into soil-filled side compartments. The ceramics was mixed with 4g Velvit Nutrilong V180 controlled release compound fertiliser (29% N, 4% P, 8% K, 1.7% MgO, C:N=0.51). This application rate was used as the ceramics contained very limited nutrients (KCl extractable; NH4 =0.16±0.06 µg gDW−1, NO3 =0.40±0.17 µg gDW−1, and NaHCO3 extractable; PO4 =0.50±0.05 µg gDW−1, n=5), and tests with different rates of application identified these concentrations as sufficient to promote strong A. capillaris germination and allow for root colonisation of side compartments. The ceramics mix was then wetted to a 1:0.65 ceramics to water ratio by weight prior to sowing. A. capillaris seeds were sown onto all mesocosm central compartments at a density of 1.8 mg cm−2 on 15th June 2023 and germinated fully within the growth chamber by 26th June 2023.

The A. capillaris was watered twice weekly with varying quantities of deionised water ranging from 50 to 500 ml depending on plant growth stage and water demands. Similar soil moisture was maintained across mesocosms and side compartments throughout the experiment (Supplementary Fig. 2). Once fully mature the A. capillaris shoots where clipped once per month or when shoots reached ≥50 cm height.

Labellinggrowthchamber
The experiment was set up and maintained in a bespoke, climate-controlled growth and continuous 13C-labelling chamber (BLOSOM)29. The chamber was maintained at 5 °C by a 4 kW FL4003 recirculating cooler (Julabo UK Ltd). Chamber air temperature was maintained at 20–22 °C by a bespoke closed-loop air conditioning system (Cambridge HOK, UK).

Grow lighting within the chamber was run on an inverted diurnal cycle with a 16 h photoperiod, using five BX120c2 and five BX180c2 LED strip lights (Valoya Ltd, Helsinki, Finland) set to 250–300 μmol m−² s−1. To achieve continuous labelling at ~500‰ δ13C, compressed air (10–30 ppm), unlabelled CO2 (99.8% pure, BOC, UK) and labelled CO2 (99% 13CO2, <2% 18O, CK isotopes Ltd, UK) controlled by precision mass flow controllers (Bronkhorst UK Ltd, Newmarket, UK) were mixed and supplied into the chamber. The average δ13C position of chamber air during the lights-on period was 506.11±26.95‰ (mean±SD).

Fluxmeasurements
CO2 flux measurements were carried out using a bespoke push-and-pull-through respiration system29 which continuously flowed CO2-free air through side compartment headspaces at ~20 ml min−1 controlled by analogue flow meters (Dwyer Instruments). During measurements the out-flow from each side compartment were connected to a Picarro G2201-i isotopic analyser (Picarro Inc. Santa Clara, CA) sequentially for 3–4 minutes until CO2 was stable.

Flux measurements of all 216 side compartments were conducted once prior to A. capillaris sowing, once prior to germination and monthly for 370 days after germination. The flux of SOM-derived CO2 from each side compartment was then derived by Eqs. (1) and (2).

Soil moisture measurements were carried out eight times throughout the experiment by inserting an IMKO HD2 Pico 32 110 mm soil moisture probe (IMKO Micromodultechnik GmbH, Germany) into the soil in each side compartment. Soil moisture calibration curves were developed for each soil independently. Reported moisture soil moisture is the average of two measurements, one at 5 cm and 10 cm soil depth.

Statisticalanalyses
All statistical analyses were performed using R version 4.3.0 (2023-04-21 ucrt). Paired t-test were performed to determine the statistical significance of differences in SOM-derived CO2 fluxes between paired side compartments with and without roots. Within group normality was confirmed by Shapiro-Wilk test. A repeated measures ANOVA was used to compare RPE ratios from active layer and permafrost soils over time. Normality of residuals was confirmed by Shapiro-Wilk test. Relationships between RPE ratio and measured soil elements were modelled using a nonlinear power function (y = ax−b) for C and N content and a linear model for C:N ratio. Models were selected by comparing goodness of fit using the Akaike information criterion (AIC). Likelihood ratio tests were performed to determine statistical significance of these models. ANCOVAs were performed to assess relative differences between active layer and permafrost soil data covarying with soil C content, N content and C:N ratio.

The concentration of SOM-derived CO2 in each side compartment headspace was calculated by the following equation:

SOM-derived CO2 (ppm) = ((δ13Cchamber(m) - δ13C SC(m)) / (δ13Cchamber(m) - δ13C soil(m))) * CO2(ppm) (1)

Where chamber is the chamber environment, SC is the side compartment headspace and soil is the soil within the side compartment.

The flux of SOM-derived CO2 from each side compartment soil was then calculated by the following equation:

SOM-derived CO2 flux (μg C gC−1 h−1) = (SOM-derived CO2 (ppm) * Flowair (ml min−1) * MCO2) / (Vm * C * 60 min h−1) (2)

Where Flowair is the incoming air flowing into the headspace, Vm is the molar volume of gas at standard temperature and pressure, MCO2 is the molar mass of 12CO2 and C is the mass of carbon in the soil sample derived from soil C content (%) and soil dry weight.

RPE ratio for each measured timepoint was then calculated by the following equation:

RPE ratio = exp(mean(ln(SOM-derived CO2 flux Roots / SOM-derived CO2 flux NoRoots))) (3)

Where SOM-derived CO2 Flux Roots is the SOM-derived CO2 flux from a side compartment with roots and SOM-derived CO2 Flux NoRoots is the SOM-derived CO2 flux from a paired side compartment without roots.

Dataavailability
All data generated for this publication are or will be available from the Environmental Information Data Centre (EIDC). Carbon and nitrogen content data from soils used in this publication can be found at https://doi.org/10.5285/54e9d93f-bacb-41e4-a66a-0cdcbdd02101. All soil flux data and soil particle size data can be found at https://doi.org/10.5285/a80c688c-0a91-41ce-a8e9-89479a38a566.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General comments\nFriggens et al. explored an interesting topic about the rhizosphere priming effects in permafrost soils, based on mesocosm\nexperiment where plants were grown in active layer and permafrost soils in a temperature-controlled environment with\ncontinuous 3CO2 labeling. The authors detected positive rhizosphere priming effects accelerated carbon release from both\nactive layer and permafrost soils, and also found the rhizosphere priming effects were associated with soil carbon and\nnitrogen content, and also soil C:N ratio. Several key issues raised during my review on this manuscript, which I think should\nbe addressed in any revised version before considering publication.\n(1) The basic background about this study should be clearly described throughout the manuscript. Particularly, how deep of\nthe permafrost layer at each sampling site and each sampling profile? How about the vertical distribution of root depth at\neach sampling site and each sampling profile? Whether could the live roots be distributed into the deep permafrost layer at\neach sampling site? Based on supplementary Table 2, it seemed like that some of permafrost samples were collected from\nvery deep layers, such as 1.5-2.0 m. It is not clear whether root growth could approach this very deep soil layer even after\npermafrost thaw. In addition, the authors hypothesized the increased rooting depth after permafrost thaw. Is there any clear\nevidence about this phenomenon? Is there any evidence to show root depth could be penetrated into the very deep\npermafrost soils examined in this study? If not, it is less meaningful to explore rhizosphere priming effects in deep permafrost\nsoils. These basic questions are fundamental for this study, should be fully addressed.\n(2) Some of experimental conditions should be better explained and greatly improved. Based on the supplementary figures\n(Figure S6-58), we can see that vegetation types varied from these sampling sites from tundra, shrub to forests. However, in\nthe mesocosm experiment, the authors only planted one species, i.e., Agrostis capillaris. Obviously, this situation could not\nreflect the in-situ conditions. Moreover, why 10 times microbial biomass N were added during the nutrient flush experiment?\nWhy was the compound fertilizer (29%N, 4%P, 8%K, 1.7%MgO, C:N = 0.51) used to simulate nutrient release upon\npermafrost thaw? In addition, there are a lot of potential variables which could affect priming effects, such as soil substrate\nquality derived from NMR analysis, and soil physico-chemical protection et al. However, this study only measured very\nsimple variables like soil carbon and nitrogen content, which meant too much other potential variables were not determined\nand ignored in this experiment. I urge the authors to take more efforts on this aspect.\n(3) The detailed experimental results for each sampling site and each sampling soil types should be clearly described.\nCurrently, the authors only showed the overall patterns of the rhizosphere priming effects for all soils and for active layer and\npermafrost soils. Given the large spatial heterogeneity among various sampling sites and sampling soil types, the readers\nare eager to know how about the rhizosphere priming effects for each sampling site and each sampling soil type. Whether\ndid the positive rhizosphere priming effects occur across all each sampling sites and each sampling soil types?\n(4) Some of results need to be better clarified, and some of discussion needs to be supported by the experimental data. For\nexample, the authors argued that rhizosphere priming ratio in active layer soils was 1.39 in days 0-185 days and 1.03\nbetween days 186-370. This transition date should be clearly shown in the related figure so that the readers could capture\nthis information very quickly. Similar issues existed in other sections where the specific date was described. In addition,\nseveral discussion parts lack data support. For example, the authors argued that rhizosphere priming effects-driven carbon\nloss may be offset by new SOM formation, but did not provide any experimental data. Similarly, those arguments about the\ndiminishing rhizosphere priming effects over time lack data support.\nSpecific comments\nLine 22: How about the results for each soil type?\nLine 29-30: It would be great to discuss the potential reasons for the stronger priming effects in permafrost than active layer\nsoils in the Discussion section.\nLine 41-43: Please provide the direct evidence to support this phenomenon, i.e., the increased rooting depth upon\npermafrost thaw.\nLine 58: The authors did not measure soil substrate quality in this study.\nLine 62: The sub-title should not occur among paragraphs within the Introduction section.\nLine 70: Several studies derived from the Tibetan permafrost regions should not be ignored. They have clearly determined\npriming effects in permafrost regions.\n(1) He M et al., 2023. Priming effect stimulates carbon release from thawed permafrost. Global Change Biology, 29: 4638-\n4651.\n(2) Chen LY, et al., 2018. Nitrogen availability regulates topsoil carbon dynamics after permafrost thaw by altering microbial\nmetabolic efficiency. Nature Communications, 9: 3951.\nLine 76: Will this situation occur definitely?\nLine 104-105: How about the results for each soil type and each sampling site?\nLine 120-123: Do these results mean that the reduced plant vigour should be the major reason? In addition, these arguments\nare purely deductive, lack direct data support. Could you add more experimental data to support these arguments?\nFigure 1 and 3: It would be better to clearly show the threshold date from whether the rhizosphere priming effects changed.\nLine 136-137: It would be better to give more explanations for this result.\nFigure 2: As mentioned in my general comments, there are a lot of potential variables which could affect priming effect. Why\nnot consider them?\nLine 174: Why 10 times microbial biomass N?\nLine 186 and line 193: It would be better to clearly show the threshold date from whether the rhizosphere priming effects\nchanged in figure 3.\nLine 215-228: These arguments need data support.\nLine 310-328: More details should be described about field sampling. For example, how many sampling sites or profiles\nwere obtained during the whole survey? How many for each soil types? What about the sampling depth for active layer and\npermafrost? How about the active layer thickness? How about the root depth distribution for each sampling site? Whether\nthe root distribution could approach the sampled permafrost soils across the various study sites?\nLine 367-369: Why was the number of mesocosms not similar? How about the potential influence?\nLine 387-389: Please give the detailed explanations why use this kind of fertilizer. Could it better chararterize the nutrient\nrelease upon permafrost thaw?\nLine 399: Please give the detailed explanations why use this species, given that vegetation types varied a lot among various\nsites.", "reviewer_2": "Nina L. Friggens and colleges have executed a really interesting and probably also laborious experiment to elucidate the\neffect of rhizodeposits on the decomposition of SOM in active-layer and permafrost soils. They find higher SOM-derived CO2\nin soils with ingrown roots than in soils without roots, and were able to attribute this increase to positive priming by 13C-\nlabelling the plants and their respiration. The authors also want to shed light on the mechanism behind the priming effect by\nadding nutrients to the soil, thereby alleviating a possible microbial N limitation and taking away the need for microbes to\nmine the SOM. Their research question is relevant and the experiment is thoughtful and looks well-executed.\nI feel the core of the experiment, i.e., the assessment of the rhizosphere priming effect in mesocosms, has been executed\nwell and the experiment is well-thought of. However, I have trouble finding the rationale behind the addition of a plant\nfertiliser (Velvit Nutrilong V180) to a soil which is probably already high in nutrients because of thawing-induced SOM\nmineralisation and then attributing the changes in priming to the alleviation of microbial N-limitation. The nutrients in the soil\nare not measured afterwards, nor is the microbial community characterised or microbial biomass measured, which are all\nkey elements possibly supporting or debunking the hypotheses stated in this paper.\nAn overall weak point of the MS is the superficial interpretation and discussion of the results. For example, the authors\ndo not elaborate on the distinction between microbial and plant N-limitation possibly steering the observed dynamics and thus\nbeing crucial for the interpretation of the results. Also they mention ‘RPEs being driven by microbial C limitation’ (r153),\nbut do not further elaborate on this finding and only discuss on N limitation further on. The short digression on SOM\nformation because of higher plant productivity should be left out in favour of a more thorough discussion and literature check of all\nfindings.\nr49/71/90: Please use rhizodeposits/rhizodeposition vs root-exudates/root-exudation consistently throughout the MS.\nr84-85: I don’t understand this sentence. How can plant-derived C be partitioned in two types of soil C?\nr103: I find the term unlabelled CO2 confusing. Without thorough reading of MM before this stage stating that plants are\ngrown entirely on labelled CO2, it could also mean root respiration or something else. I suggest to clarify this term before, or\nuse something more specific such as SOM-derived CO2.\nr108: Supplementary fig. 2 is not referred to anywhere in the MS.\nr108-109: I’m struggling with the time scales of this experiment. Root exudates are labile C-components, so when root\nexudates are halted because of severing plant roots, you would expect a much faster reaction of microbial respiration than\n‘within a month’, even if soil temperature was only 5°C in your experiment. See eg. (Müller et al., 2022), where these kind of\ndynamics change overnight. Why did you only measure CO2 concentration a month after severing roots?\nFigure 1&3: I’m not too fond of two-axes figures because they are confusing and unnecessary; the scales of the axes are\ndifferent and the units are not comparable, standard error bars and 95% confidence bars are used in the same window. But\nof course, this is more of a formatting issue.\nFigure 2: The relationship between %C, %N and RPE ratio looks hardly linear. It seems that RPE ratio is higher when %C <\n5 and %N < .5 or so, and then there is a flat line when %C is between 5 & 40%, and N is between .5 and 3%. Given the\nmuch larger range of the flat line and the fact that you are also comparing active layer and permafrost soils, this is not trivial.\nA boxplot showing %C and %N categories, and analysis with an ANOVA would statistically be more sound.\nr164: if p=0.24, how is this statistically significant?\nr188-189: ‘may alleviate RPEs’: What do you mean with this? One could understand ‘may alleviate microbial N-limitation,\nleading to lower N-mining in SOM and resulting in the absence of a priming effect’. However, more likely than a microbial N\nlimitation, especially in soils that had a thaw-related nutrient flush, is a plant N limitation and a microbial C limitation, which\nis explained nicely in this paper (Soong et al., 2020). This assumption would lead to an entirely different interpretation of the\nresults. Moreover, no evidence or measurements are given for the statement that the available nutrient pool diminished after\n185 days. The initial absence of RPE and recovery later on might be induced by other dynamics as well, like a N-induced\nshift in microbial community rather than RPEs being driven by nutrient demand.\nr215-228: It is true that little is known about SOM formation and stabilisation in high-latitude areas, but this is not what your\nexperiment or research is about. Maybe the MS would benefit more from a deeper interpretation of your (interesting!) results\nthan from this digression.\nr459-469: Did you test your data for within-group normality (t-test) or normality of the residuals (ANOVA). It’s not mentioned\nhere, but remains an important assumption.\nr475: Please have a look at the notation of this equation. Mention units consistently (60 min h-1), mend spaces (mlmol-1),\nwrite physical quantities and units the same for each term (g C), and take in account conversions (g → µg)\nr476: Why FLOWair is capitalised when it’s not in the equation?\nr480: I don’t understand why the RPE ratio is noted like exp(ln(CO2fluxroots/CO2fluxnoroots)), which just comes down to\nCO2fluxroots/CO2fluxnoroots for values other than 0.\nSupplementary\nr24: moth → month\nMüller, R., Maier, A., Inselsbacher, E., Peticzka, R., Wang, G., & Glatzel, S. (2022). 13C-Labeled Artificial Root Exudates Are\nImmediately Respired in a Peat Mesocosm Study. Diversity, 14(9), Article 9. https://doi.org/10.3390/d14090735\nSoong, J. L., Fuchslueger, L., Marañon-Jimenez, S., Torn, M. S., Janssens, I. A., Penuelas, J., & Richter, A. (2020). Microbial\ncarbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling. Global\nChange Biology, 26(4), 1953–1961. https://doi.org/10.1111/gcb.14962"}, "author_response": "We thank the reviewer for highlighting that more detail was required here. To address the\nreviewer’s concerns we have added text detailing the observed presence of roots and how\nthis indicates downward root growth in response to active-layer deepening in the introduction\n(L55-60) and in greater detail in the soil sampling part of the methods section (L473-478).\nAdditionally, we have added active-layer thickness information to supplementary table 2.\nAdditionally, please find a detailed response to the reviewer’s specific questions below:\nThe occurrence of dead roots within epigenetic permafrost is widespread along the western\nArctic coast, Canada, and reflects plant growth in a deeper-than-present active layer during\nthe early Holocene (Burn, 1997). Later active-layer thinning froze the roots within permafrost.\nThe base of the deepened active layer is marked by a thaw unconformity in the permafrost\nstratigraphy. Radiocarbon dating of roots and rhizomes in the basal part of the palaeo-active\nlayer indicates that the maximum development of the active layer occurred about 11,000–\n9,000 calendar years ago (Burn, 1997; Murton et al., 1998). ALT in the Tuktoyaktuk\nCoastlands, where profiles P1 to P6 were sampled, at that time was about 2.5 times thicker\nthan present largely because the warmer climate allowed development of forest, which\ntrapped snow and increased ground temperatures in winter (Burn, 1997). Overall, therefore it\nis clear that roots grew downward through newly thawed soil in a deepening active layer\nduring the last major episode of global warming during the last glacial-to-interglacial\ntransition prior to refreezing within aggrading permafrost in the mid to late Holocene. Finally,\nwhile setting up the experiment we found that, while roots were more abundant in active\nlayer samples, we can confirm root presence in all permafrost soils.\nBurn CR. 1997. Cryostratigraphy, paleogeography, and climate change during the early\nHolocene warm interval, western Arctic coast, Canada. Canadian Journal of Earth Sciences\n34, 912–925.\nMurton JB, French HM, Lamothe M. 1998. The dating of thermokarst terrain, Pleistocene\nMackenzie Delta, Canada. In: Lewkowicz AG, Allard M. (eds) Proceedings of the\n7th International Permafrost Conference, Yellowknife, Canada, 23–27 June 1998, Collection\nNordicana, Centre d’études nordiques, Université Laval, pp. 777–782.\nMurton JB, Goslar T, Edwards ME, Bateman MD, Danilov PP, Savvinov GN, Gubin SV,\nGubin SV, Ghaleb B, Haile J, Kanevskiy M, Lozhkin AV, Lupachev AV, Murton DK, Shur Y, Tikhonov A,\nVasil’chuk AC, Vasil’chuk YK, Wolfe SA. 2015. Palaeoenvironmental interpretation of\nyedoma silt (ice complex) deposition as cold-climate loess, Duvanny Yar, northeast Siberia.\nPermafrost and Periglacial Processes 26, 207–288.\n(2) Some of experimental conditions should be better explained and greatly improved. Based\non the supplementary figures (Figure S6-58), we can see that vegetation types varied from\nthese sampling sites from tundra, shrub to forests. However, in the mesocosm experiment,\nthe authors only planted one species, i.e., Agrostis capillaris. Obviously, this situation could\nnot reflect the in-situ conditions. Moreover, why 10 times microbial biomass N were added\nduring the nutrient flush experiment? Why was the compound fertilizer (29%N, 4%P, 8%K,\n1.7%MgO, C:N = 0.51) used to simulate nutrient release upon permafrost thaw? In addition,\nthere are a lot of potential variables which could affect priming effects, such as soil substrate\nquality derived from NMR analysis, and soil physico-chemical protection et al. However, this\nstudy only measured very simple variables like soil carbon and nitrogen content, which\nmeant too much other potential variables were not determined and ignored in this\nexperiment. I urge the authors to take more efforts on this aspect.\nThese are all fair points, and we are glad to have to opportunity to clarify. It is highly\nchallenging/impossible to simulate field conditions fully in the laboratory, and we believe our\napproach is the correct balance between realism and what is experimentally possible, and\ngoes substantially beyond previous studies in this regard. Firstly, it would be very difficult to\nrecreate tundra and northern boreal plant communities in the control growth chamber system\nat the scales used. Therefore, Agrostis capillaris. was considerd the ideal plant species as it\ncombines a distribution that includes high northern latitudes with high germination rates from\nseed and an ability to establish and grow well in growth chamber studies. Text further\nexplaining this has been added to the manuscript (L554-556).\nTen times microbial biomass N were added to soils to simulate a thaw-related nutrient flush.\nThis level of nutrient addition was chosen to experimentally push the soil system into a state\nof nutrient abundance which would last for an extended time period even in the presence of\na large nutrient sink namely the actively growing plants. This is also why a compound\nfertiliser specifically designed to release nutrients over a long period of time was used. The\nuse of a fertiliser containing multiple macro nutrients was deemed to more accurately reflect\nthe range of nutrients released as a result of permafrost thaw (Finger et al. 2016; Yang et al.\n2021), as opposed to a pure N addition often used in these types of experiments. Text\nfurther explaining this has been added to the manuscript (L301-305 & L539-541).\nIt is true that many potential variables may influence soil priming effects, and it will be\ninteresting to explore in future work building on the current study. Our approach to identifying\ncontrols on priming effects is consistent with other high-latitude and global synthesis studies\nwhich have considered organic matter contents and C:N ratios as dominant drivers of\ndecomposition rates (e.g. Schadel et al GCB). We have included the major permafrost soil\ntypes represent in this region. To highlight this further to the reader we have added ‘Soil type’\nas a shape variable to Figures 2 & 4 in the manuscript. In addition to this we have expanded\nSupplementary Figure 1 to include plots examining the relationship between RPEs and\ncumulative SOM-derived flux (as a proxy for SOM bioavailability) and primary texture\nvariables. We also identify priorities for future work that reflect the very valid suggestions\nfrom this reviewer.\nFinger RA, Turetsky MR, Kielland K, et al (2016) Effects of permafrost thaw on nitrogen\navailability and plant–soil interactions in a boreal Alaskan lowland. Journal of Ecology\n104:1542–1554. https://doi.org/10.1111/1365-2745.12639\nYang G, Peng Y, Abbott BW, et al (2021) Phosphorus rather than nitrogen regulates\necosystem carbon dynamics after permafrost thaw. Global Change Biology 27:5818–\n5830. https://doi.org/10.1111/gcb.15845\n(3) The detailed experimental results for each sampling site and each sampling soil types\nshould be clearly described. Currently, the authors only showed the overall patterns of the\nrhizosphere priming effects for all soils and for active layer and permafrost soils. Given the\nlarge spatial heterogeneity among various sampling sites and sampling soil types, the\nreaders are eager to know how about the rhizosphere priming effects for each sampling site\nand each sampling soil type. Whether did the positive rhizosphere priming effects occur\nacross all each sampling sites and each sampling soil types?\nThanks for this suggestion. In figures 2 and 4 in the manuscript we have included\ninformation on RPE ratio split by soil type denoted by the shape of the points in these plots.\nThis will help the reader see that there are no overall patterns in RPE ratios by soil type both\nwith and without nutrient addition. We are unable to explore relationships between RPE\nratios and sampling sites as our experimental design only replicates soil types, not sampling\nsampling\nsites.\n(4) Some of results need to be better clarified, and some of discussion needs to be\nsupported by the experimental data. For example, the authors argued that rhizosphere\npriming ratio in active layer soils was 1.39 in days 0-185 days and 1.03 between days 186-\n370. This transition date should be clearly shown in the related figure so that the readers\ncould capture this information very quickly. Similar issues existed in other sections where the\nspecific date was described. In addition, several discussion parts lack data support. For\nexample, the authors argued that rhizosphere priming effects-driven carbon loss may be\noffset by new SOM formation, but did not provide any experimental data. Similarly, those\narguments about the diminishing rhizosphere priming effects over time lack data support.\nAs requested, we have generated the plot with edits which denote the durations over which\nwe summarise “priming” and “no priming” using variations in the plot background colour. We\nhave included the modified plots below.\nWhilst we see the reviewer’s point about enabling the reader to capture information quickly,\nwe feel that the requested edits to the plots only make them busier and harder to interpret at\na glance, but if the reviewer disagrees, we can update the graphs in the manuscript to the\nones included below. To improve the readability of Figures 1 & 3 in the manuscript (and in\nresponse to a suggestion from Reviewer 2) we have simplified the plots, removed the\nsecondary y-axis and plotted RPE ratio and SOM-derived CO flux separately for all soils\n2\nand each horizon.\nIn response to a comment by reviewer 2 the section on the potential for RPE-driven carbon\nloss to be offset by new SOM formation has been removed in order to place more emphasis\non experimental data.\nSpecific comments\nLine 22: How about the results for each soil type?\nIn figures 2 and 4 in the manuscript we have included information on RPE ratio split by soil\ntype denoted by the shape of the points in these plots. This will help the reader see that\nthere are no overall patterns in RPE ratios by soil type both with and without nutrient\naddition. \nLine 29-30: It would be great to discuss the potential reasons for the stronger priming effects\nin permafrost than active layer soils in the Discussion section.\nWe have added additional text further discussing the stronger priming effects in permafrost\nthan active layer soils (L200-238). Reviewer 2 has requested more detailed and in-depth\ndiscussion of the experimental results presented here. In order to address both reviewers’\nrequests we have added additional detail and discussion of results throughout the\nmanuscript. We do not include a specific discussion section (as permitted by the journal\nformatting guidelines), as we feel the results lend themselves to be discussed as they arise\nin this case.\nLine 41-43: Please provide the direct evidence to support this phenomenon, i.e., the\nincreased rooting depth upon permafrost thaw.\nAdditional text has been added to the introduction (L55-60) and soil sampling part of the\nmethods section (L473-478) to provide additional support for the statements regarding\nincreased rooting depth upon permafrost thaw. See also response to general comment 1\nabove.\nLine 58: The authors did not measure soil substrate quality in this study.\nThe reviewer is correct that substrate quality was not measured in this study. However, the\npoint we make in the introduction is a theoretical one highlighting that SOM quality may\naffect soil priming and we therefore feel it remains relevant to the overall point being made.\nIn addition to this we have expanded Figure 2 to include plots examining the relationship\nbetween RPEs and cumulative SOM-derived CO flux as a proxy for SOM bioavailability or\nLine 62: The sub-title should not occur among paragraphs within the Introduction section.\nSubheadings have been removed from the introduction as per the formatting requirements.\nLine 70: Several studies derived from the Tibetan permafrost regions should not be ignored.\nThey have clearly determined priming effects in permafrost regions.\n(1) He M et al., 2023. Priming effect stimulates carbon release from thawed permafrost.\nGlobal Change Biology, 29: 4638-4651.\n(2) Chen LY, et al., 2018. Nitrogen availability regulates topsoil carbon dynamics after\npermafrost thaw by altering microbial metabolic efficiency. Nature Communications, 9: 3951.\nWe thank the reviewer for the suggested literature. We have read and cited these where\nrelevant (L216-218, L253-257, L275-277, L322-326).\nLine 76: Will this situation occur definitely?\nAdditional text has been added to the introduction (L55-60) and soil sampling part of the\nmethods section (L473-478) to provide additional support for the statements regarding\nincreased rooting depth upon permafrost thaw. See also response to general comment 1\nabove.\nLine 104-105: How about the results for each soil type and each sampling site?\nIn figures 2 and 4 in the manuscript we have included information on RPE ratio split by soil\ntype denoted by the shape of the points in these plots. This will help the reader see that\nthere are no overall patterns in RPE ratios by soil type both with and without nutrient\naddition. We are unable to explore relationships between RPE ratios and sampling sites as\nour experimental design only replicates soil types, not sampling sites.\nLine 120-123: Do these results mean that the reduced plant vigour should be the major\nreason? In addition, these arguments are purely deductive, lack direct data support. Could\nyou add more experimental data to support these arguments?\nEven though above-ground growth rates declined and the plants required less frequent\nclipping as the experiment went on, the headspace CO concentration remained similar\n2\nthroughout the experiment (Supplementary Fig. 3a) and did not decline over time,\nsuggesting that similar levels of root activity were maintained throughout the duration of the\nexperiment. We have edited the text to better explaining this (L174-180).\nFigure 1 and 3: It would be better to clearly show the threshold date from whether the\nrhizosphere priming effects changed.\nPlease see the response to general comment 4 where we show plots with the change in\npriming effects denoted.\nLine 136-137: It would be better to give more explanations for this result.\nWe have added additional text further discussing the stronger priming effects in permafrost\nthan active-layer soils (L201-234).\nFigure 2: As mentioned in my general comments, there are a lot of potential variables which\ncould affect priming effect. Why not consider them?\nThe reviewer makes a good point about the many potential variables which may influence\nsoil priming effects and it will be interesting to explore in future work building on the current\nstudy. In the current work we sought to test hypotheses linked to nutrient mining which are\nhypothesised to be particularly important for high-latitude soils. To address the points made\nby the reviewer here and in the general comments, we have expanded Figure 2 to include\nplots examining the relationship between RPEs and cumulative SOM-derived CO flux (as a\n2\nproxy for SOM bioavailability) and Supplementary Figure 1 has been expanded to include\nrelationships between RPEs and primary texture variables.\nLine 174: Why 10 times microbial biomass N?\nTen times microbial biomass N was added to soils to simulate a large level of thaw-related\nnutrient flush, and to reduce or eliminate microbial nutrient limitation. This is why the level of\napplication was made proportional to microbial biomass N and was chosen to experimentally\npush the soil system into a state of nutrient abundance which would persist for an extended\nduration. This is also why a compound fertiliser specifically designed to release nutrients\nover a long period of time was used. Text further explaining this has been added to the\nmanuscript (L302-306 and L545-546).\nLine 186 and line 193: It would be better to clearly show the threshold date from whether the\nrhizosphere priming effects changed in figure 3.\nPlease see the response to general comment 4 where we show plots with the change in\npriming effects denoted.\nLine 215-228: These arguments need data support.\nIn response to a comment by reviewer 2 the section on the potential for RPE-driven carbon\nloss to be offset by new SOM formation has been removed in order to place more emphasis\non experimental data.\nLine 310-328: More details should be described about field sampling. For example, how\nmany sampling sites or profiles were obtained during the whole survey? How many for each\nsoil types? What about the sampling depth for active layer and permafrost? How about the\nactive layer thickness? How about the root depth distribution for each sampling site?\nWhether the root distribution could approach the sampled permafrost soils across the\nvarious study sites?\nThe field sampling programme collected soil samples from vertical profiles at 14 sites.\nLimitations in sample size and available space in the growth chamber prevented samples\nfrom two Orthels beside the East Channel of the Mackenzie River from being analysed.\nThe field sampling programme and the presence of roots in the sampled profiles has been\ndescribed in greater detail in the soil sampling part of the methods section (L4551-478).\nAdditionally, we have added further active-layer thickness information to supplementary table\n2.\nLine 367-369: Why was the number of mesocosms not similar? How about the potential\ninfluence?\nIn the design, we maximised the number of independent soil types that we studied, providing\nconfidence in the priming results across contrasting permafrost soils. The nutrient\nmanipulation experiment was included to add further mechanistic understanding, and we\nmeasured as many soil types as possible within size constraints imposed by the labelling\nchamber. We do not make direct statistical comparisons between these two experiments.\nWe chose to carefully distribute the available space and soil samples in a way which\nenabled the greatest range of soil types, horizons, C and N contents to be analysed within\nthe physical limitations of our experimental set up and available soils.\nThe statistical effects of the uneven number were checked and found to have no significant\neffect. Text has been added to the methods section (L519-520) to make this more explicit for\nthe reader.\nLine 387-389: Please give the detailed explanations why use this kind of fertilizer. Could it\nbetter chararterize the nutrient release upon permafrost thaw?\nThe use of a fertiliser containing multiple macro nutrients was deemed to more accurately\nreflect the effects of a thaw-induced nutrient flush (Finger et al. 2016; Yang et al. 2021),\nas opposed to a pure N addition often used in these types of experiments. A compound fertiliser\nspecifically designed to release nutrients over a long period of time was used so that the\nfertilisation effect would last for the duration of the experiment. Text further explaining this\nhas been added to the manuscript (L301-305 & L539-541). See also response to general\ncomment 2.\nLine 399: Please give the detailed explanations why use this species, given that vegetation\ntypes varied a lot among various sites.\nAgrostis capillaris was used because it combines a distribution that includes high northern\nlatitudes with high germination rates from seed and an ability to establish and grow well in\ngrowth chamber studies. Therefore, this species is relevant to the ecosystems involved and\nexperimentally tractable. Including the varied vegetation types is not possible given the size\nof the bespoke growth chamber to house trees and shrubs. Therefore, the pragmatic\napproach was to use a single, widespread species that was experimentally tractable. We\nargue that our approach still represents a major advance over priming studies that use 13C-\nlabelled substrates without live plants, while also recognising that no lab experiment can fully\nreplicate field conditions. Text further explaining this has been added to the manuscript\n(L554-560). Future studies that investigate the extent to which plant species or functional\ntype affect priming rates in permafrost soils would be very valuable but were beyond the\nscope of this study.\nWe thank the reviewer for a constructive and helpful review which has greatly improved the\nmanuscript. Furthermore, we appreciate that whilst the reviewer is rightly critical of some\nelements of the experiment and manuscipt, they acknowledge how much work has gone into\nexecuting this piece of experimental work.\nThe rationale to use high levels of a slow-release compound fertiliser was threefold: a) to\npush the soil microbial community into a state of nutrient abundance even in the presence of\na large nutrient sink namely the actively growing plants, thus removing the microbial nutrient\nlimitation to aid testing our microbial nutrient demand hypothesis for RPEs, b) to introduce a\nrange of nutrients as opposed to simply N, and c) to enable the slow and even release of\nnutrients throughout the long-term experiment. We have added text to the manuscript\nexplaining this (L301-306, L539-541 & L545-546).\nThe reviewer makes a good point about measuring the soil nutrients, microbial community\nand biomass; this would likely have improved the strength of the deductions made about\nmicrobial nutrient limitations. However, it would have required continuous destructive\nsampling. While there is a flush of nutrients on thaw the strong plant growth and plant\nnutrient uptake necessitates the need for additional nutrient release to reduce microbial\nnutrient limitation. We take the reviewer’s point that we have contextualised the discussion\naround microbial nutrient limitation, but now also discuss the results from the perspective of\nplant nutrient limitation as well (L280-287), recognising we cannot necessarily distinguish\nbetween them.\nThroughout the manuscript we have expanded the discussion of the results presented,\nadding more details, thorough interrogation, discussion and contextualisation of observed\npatterns in our data. We believe that this has greatly improved the manuscript, and thank the\nreviewer again for their suggestions.\nr49/71/90: Please use rhizodeposits/rhizodeposition vs root-exudates/root-exudation\nconsistently throughout the MS.\nAll changed to consistently be termed ‘root exudates/exudation’ in text.\nr84-85: I don’t understand this sentence. How can plant-derived C be partitioned in two types\nof soil C?\nHere we mean that soil C fluxes can be partitioned into plant-derived C and RPE-induced\nrelease of soil C. We have clarified the sentence in text to:\n“The use of 13CO and soils with and without roots allows partitioning of C effluxes from soil\n2\ninto i) plant-derived C and ii) RPE-induced release of soil C.” (L112-114)\nr103: I find the term unlabelled CO2 confusing. Without thorough reading of MM before this\nstage stating that plants are grown entirely on labelled CO2, it could also mean root\nrespiration or something else. I suggest to clarify this term before, or use something more\nspecific such as SOM-derived CO2.\nWe like the reviewer’s suggestion of using the term ‘SOM-derived CO ’ in place of\n2\n‘Unlabelled CO ’ as this is much clearer. We have changed the term in all relevant places in\n2\nthe manuscript text and figures.\nr108: Supplementary fig. 2 is not referred to anywhere in the MS.\nWe have now added text including reference to Supplementary figure 2 to the methods\nsection of the paper (L578-580).\nr108-109: I’m struggling with the time scales of this experiment. Root exudates are labile C-\ncomponents, so when root exudates are halted because of severing plant roots, you would\nexpect a much faster reaction of microbial respiration than ‘within a month’, even if soil\ntemperature was only 5°C in your experiment. See eg. (Müller et al., 2022), where these\nkind of dynamics change overnight. Why did you only measure CO2 concentration a month\nafter severing roots?\nThe reviewer is correct in pointing out that the drop in SOM-derived CO flux likely occurred\n2\nsooner than one month as root exudates are turned over and respired within days, therefore\nthe effect on SOM-derived CO fluxes is likely short lived. We were unable to capture this\n2\nempirically in this experiment as fluxes were monitored on average once per month, which\nstill represented a considerable amount of work as the reviewer highlights. It would have\nbeen valuable data to have; however, without them we can only say for certain when the\ndrop in SOM-derived CO flux occurred within the month. We would argue that this does not\n2\ndistract from any of our evidence of priming, we just don't have the temporal resolution to\ndetect this rapid change. Text explaining this has been added to the manuscript (L150-155).\nFigure 1&3: I’m not too fond of two-axes figures because they are confusing and\nunnecessary; the scales of the axes are different and the units are not comparable, standard\nerror bars and 95% confidence bars are used in the same window. But of course, this is\nmore of a formatting issue.\nWe have split the double axes plots in question into two separate single axes plots each but\nmaintained the layout and common x-axis so that the RPE ratio and SOM-derived CO\n2\nfluxes can be interpreted on the same x-axis (Figs 1 & 3).\nFigure 2: The relationship between %C, %N and RPE ratio looks hardly linear. It seems that\nRPE ratio is higher when %C < 5 and %N < .5 or so, and then there is a flat line when %C is\nbetween 5 & 40%, and N is between .5 and 3%. Given the much larger range of the flat line\nand the fact that you are also comparing active layer and permafrost soils, this is not trivial. A\nboxplot showing %C and %N categories, and analysis with an ANOVA would statistically be\nmore sound.\nWe thank the review for the suggestion to look at the relationship between %C, %N and\nRPE ratio and consider whether linear models are appropriate. Upon re-examining these\nrelationships using non-linear models we have found that a power function model is the best\nfit for the %C and %N data, while a linear model remains the best fit for the C:N data. These\nmodels were selected by comparing goodness of fit using the Akaike information criterion\n(AIC). We have updated all relevant plots and text in both the results (L246-253) and\nmethods (L622-625) sections to reflect this.\nWe feel that using non-linear models for these continuous %C and %N data is more\nappropriate than creating boxplots of artificially selected bins for comparison. If the reviewer\ndoes not agree with this way of handling these data, we are happy to provide the suggested\nbox plot but identifying data bins is somewhat subjective, and would result in different\nnumbers of observations in different bins. We hope the reviewer agrees with our decision to\nbase the analysis on the full dataset.\nr164: if p=0.24, how is this statistically significant?\nThis was a typo. After re-running the statistical tests and double checking the outputs it turns\nout that the RPE ratio was significantly positively related to C:N ratio in active-layer soils\n(R2=0.38, p=0.024). We have corrected this error in the manuscript text.\nr188-189: ‘may alleviate RPEs’: What do you mean with this? One could understand ‘may\nalleviate microbial N-limitation, leading to lower N-mining in SOM and resulting in the\nabsence of a priming effect’. However, more likely than a microbial N limitation, especially in\nsoils that had a thaw-related nutrient flush, is a plant N limitation and a microbial C limitation,\nwhich is explained nicely in this paper (Soong et al., 2020). This assumption would lead to\nan entirely different interpretation of the results. Moreover, no evidence or measurements\nare given for the statement that the available nutrient pool diminished after 185 days. The\ninitial absence of RPE and recovery later on might be induced by other dynamics as well,\nlike a N-induced shift in microbial community rather than RPEs being driven by nutrient\ndemand.\nThe reviewer makes some excellent points here. We have expanded this section of the\nmanuscript to include some of these suggestions as well as improve upon the discussion of\nthe nutrient addition data and how this links to the data from soils without nutrient addition\n(L316-358).\nr215-228: It is true that little is known about SOM formation and stabilisation in high-latitude\nareas, but this is not what your experiment or research is about. Maybe the MS would\nbenefit more from a deeper interpretation of your (interesting!) results than from this\ndigression.\nWe have removed this section from the manuscript as per the reviewer’s suggestion.\nr459-469: Did you test your data for within-group normality (t-test) or normality of the\nresiduals (ANOVA). It’s not mentioned here, but remains an important assumption.\nWithin group and residual normality was confirmed using the Shapiro-Wilk test. Text stating\nthis has been added to the methods section (L621-622).\nr475: Please have a look at the notation of this equation. Mention units consistently (60 min\nh-1), mend spaces (mlmol-1), write physical quantities and units the same for each term (g\nC), and take in account conversions (g → µg)\nWe have double-checked the notation of the equation in question and made relevant edits\nas necessary (L636-639).\nr476: Why FLOWair is capitalised when it’s not in the equation?\nThis error has been corrected in the text, so that the equation and text now match (L640).\nr480: I don’t understand why the RPE ratio is noted like\nexp(ln(CO2fluxroots/CO2fluxnoroots)), which just comes down to\nCO2fluxroots/CO2fluxnoroots for values other than 0.\nThis was indeed not very clear in the original text. We have now clarified in the manuscript\n(L646) that the RPE ratio for each measured time point was calculated by:\nexp(mean(ln(CO2fluxroots/CO2fluxnoroots))). It is essential to use natural logs when\naveraging ratios, otherwise average ratios greater than one are likely to be observed even\nwhen there is no overall response. This is analogous to the approach used in meta analyses.\nSupplementary\nr24: moth → month\nCorrected in text.\nMüller, R., Maier, A., Inselsbacher, E., Peticzka, R., Wang, G., & Glatzel, S. (2022). 13C-\nLabeled Artificial Root Exudates Are Immediately Respired in a Peat Mesocosm Study.\nDiversity, 14(9), Article 9. https://doi.org/10.3390/d14090735\nSoong, J. L., Fuchslueger, L., Marañon-Jimenez, S., Torn, M. S., Janssens, I. A., Penuelas,\nJ., & Richter, A. (2020). Microbial carbon limitation: The need for integrating microorganisms\ninto our understanding of ecosystem carbon cycling. Global Change Biology, 26(4), 1953–\n1961. https://doi.org/10.1111/gcb.14962\nWe thank both reviewers for their helpful comments and final revisions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the authors for their great efforts to address my previous comments. Compared with earlier studies available in permafrost regions, this study added valuable insights into priming effects by conducting a long-term labeling mesocosm experiment (with real plants，climate-controlled growth and continuous C13 labeling chamber experiment). Several remaining minor points need to be addressed.\n(1) Line 101: “Results” section should be replaced with “Results and discussion”, since the authors also discussed the related results in this section.\n(2) Line 110: To improve the readability, it would be better to briefly mention the labeling mesocosm experiment before the detailed description of the results，given the detailed methods occurred at the end of the paper.\n(3) Figure 1 and the corresponding results section: Based on this figure, we can see that the priming effect was significant in only two incubation days for the active layer (Fig.1c). Similarly, the priming effect was not significant for all of incubation days in the permafrost (Fig.1c). The authors needed to clarify this point and discuss more for this issue.\n(4) Line 170: This argument could not be obtained directly from Fig.2d. It would be better to add an inserted panel for showing this comparison between permafrost and active layer.\n(5) Line 235: the reduction of microbial nutrient limitation.\n(6) Line 247-248: Could the authors conduct some kind of statistical comparison to illustrate this point in a more direct manner?", "reviewer_2": "Nina L. Friggens and colleagues have edited their manuscript thoroughly and made an effort to respond to my comments.\nMost of my questions or comments have been well addressed, although some experimental issues cannot be dealt with without repeating the experiment, such as the low temporal resolution of the sampling, or the lack of destructive sampling to get more insight into soil nutrient dynamics, microbial biomass or community. Although I still consider this to be a weakness of the manuscript, which inevitably leads to speculation in the discussion of plant or microbial nutrient status and what exactly causes the RPE, I agree with the authors in their reasoning that the lack of this information does not necessarily weaken their main conclusion. They have also added, as I requested, a better contextualisation of their results in the discussion, and they also now have a very good, relevant and important conclusion.\nr307: Here you mention N-mining as a plant-driven process, but it's a microbial process. Also, microbial N-mining causing positive priming (r54: 'This implies that RPEs are controlled by microbial nutrient demand') is controversial and has been challenged in the literature, so this statement should be nuanced and positive priming should not be attributed to N-mining without reservation (Verbrigghe et al., 2022; Wild et al., 2019).\nVerbrigghe, N., Meeran, K., Bahn, M., Fuchslueger, L., Janssens, I. A., Richter, A., Sigurdsson, B. D., Soong, J. L., & Vicca, S. (2022). Negative priming of soil organic matter following long-term in situ warming of sub-arctic soils. Geoderma, 410, 115652. https://doi.org/10.1016/j.geoderma.2021.115652\nWild, B., Li, J., Pihlblad, J., Bengtson, P., & Rütting, T. (2019). Decoupling of priming and microbial N mining during a short-term soil incubation. Soil Biology and Biochemistry, 129, 71–79. https://doi.org/10.1016/j.soilbio.2018.11.014"}, "author_response": "We have changed this in the manuscript text.\n(2) Line 110: To improve the readability, it would be better to briefly mention the labeling\nmesocosm experiment before the detailed description of the results，given the detailed\nmethods occurred at the end of the paper.\nWe thank the reviewer for this suggestion and have added the following text (L109-113):\n“The collected permafrost and active layer soils were used to investigate rhizosphere priming\neffects in permafrost region soils within a temperature controlled, 13C-labelling plant-growth\nchamber27. By measuring soil C effluxes from the headspace of both rooted and root-free\ncompartments in custom-designed mesocosms (see Methods for details), we were able to\npartition C effluxes into plant-derived C and RPE-induced release of soil C.”\n(3) Figure 1 and the corresponding results section: Based on this figure, we can see that the\npriming effect was significant in only two incubation days for the active layer (Fig.1c).\nSimilarly, the priming effect was not significant for all of incubation days in the permafrost\n(Fig.1c). The authors needed to clarify this point and discuss more for this issue.\nText to emphasise this has been added in L137-138 and L151-152.\n(4) Line 170: This argument could not be obtained directly from Fig.2d. It would be better to\nadd an inserted panel for showing this comparison between permafrost and active layer.\nWe have added an insert to Figure 2d as per the reviewer’s suggestion.\n(5) Line 235: the reduction of microbial nutrient limitation.\nThis has been corrected in the manuscript text.\n(6) Line 247-248: Could the authors conduct some kind of statistical comparison to illustrate\nthis point in a more direct manner?\nWe have performed a paired t-test and amended the relevant text to include the results.\nReviewer #2 (Remarks to the Author):\nNina L. Friggens and colleagues have edited their manuscript thoroughly and made an effort\nto respond to my comments. Most of my questions or comments have been well addressed,\nalthough some experimental issues cannot be dealt with without repeating the experiment,\nsuch as the low temporal resolution of the sampling, or the lack of destructive sampling to\nget more insight into soil nutrient dynamics, microbial biomass or community. Although I still\nconsider this to be a weakness of the manuscript, which inevitably leads to speculation in the\ndiscussion of plant or microbial nutrient status and what exactly causes the RPE, I agree\nwith the authors in their reasoning that the lack of this information does not necessarily\nweaken their main conclusion. They have also added, as I requested, a better\ncontextualisation of their results in the discussion, and they also now have a very good,\nrelevant and important conclusion.\nr307: Here you mention N-mining as a plant-driven process, but it's a microbial process.\nAlso, microbial N-mining causing positive priming (r54: 'This implies that RPEs are controlled\nby microbial nutrient demand') is controversial and has been challenged in the literature,\nso this statement should be nuanced and positive priming should not be attributed to N-mining\nwithout reservation (Verbrigghe et al., 2022; Wild et al., 2019).\nr307: We have amended the manuscript text to avoid suggesting that N-mining is a plant-\ndriven process.\nr54: We thank the reviewer for the suggested references which do indeed add nuance to the\ncontext of the role of N-mining in positive priming effects. We have amended the text in L53-\n54 to read; “This implies that RPEs may be controlled by microbial nutrient demand,\nhowever evidence from incubation experiments suggests that microbial N-mining may not be\na key driver of positive priming.”\nVerbrigghe, N., Meeran, K., Bahn, M., Fuchslueger, L., Janssens, I. A., Richter, A.,\nSigurdsson, B. D., Soong, J. L., & Vicca, S. (2022). Negative priming of soil organic matter\nfollowing long-term in situ warming of sub-arctic soils. Geoderma, 410,\n115652. https://doi.org/10.1016/j.geoderma.2021.115652\nWild, B., Li, J., Pihlblad, J., Bengtson, P., & Rütting, T. (2019). Decoupling of priming and\nmicrobial N mining during a short-term soil incubation. Soil Biology and Biochemistry, 129,\n71–79. https://doi.org/10.1016/j.soilbio.2018.11.014"}]

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