Warming Does Not Change Vertical Variations in Microbial Resource Limitation in Subtropical Forests at China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

26 forests-3420102 -peer-review-v1

 REVIEW

Dear authors,

The article is devoted to the change in the physicochemical, microbiological and enzymatic characteristics of soils along the profile up to 60 cm in the control soil and in soil with an artificial increase heating by 4 ° C in the tropical forests of China. The article is important and informative, but there are the following comments below.

General comment on the entire text: The authors constantly and frequently use the word limitation throughout the article, and it is often unclear what this word means. The authors should write in the text what they mean by the word limitation. It is better to replace the word limitation in some cases throughout the text with other words, so that it is clearer what is meant - an increase or decrease in the amount of a particular indicator along the profile.

302 For example,  4. Discussion In this study, we provided experimental evidence for the response of soil microbial 302 resource limitation to whole-soil-profile warming and the associated mechanisms in sub-303 tropical forests (Figure 6). We observed that microbial nutrient (N and P) limitation (replace with content) in-304 creased but microbial C limitation decreased consistently along the soil profile regardless 305 of warming. We also found that the vertical variations in microbial N limitation, etc.

Add Title: Warming does not change vertical variations in microbial re-source limitation in subtropical forests at China

In the  Abstract:   F/B - you need to decipher what this abbreviation means

In the  Abstract: Need Describe the experiment, objects, and methods. Where the object is located, what soil layers and to what depth were studied, what kind of warming was created, etc.

Need Significantly shorten the captions in all figures. They are often repeated in all figures. and give an explanation of all the abbreviations in all the figures

 

 Abstract:  17. microbial nutrient (nitrogen and phosphorus) limitation This it is incorrect to write, because nitrogen and phosphorus are also needed by plants, not just by microbes.

17-18. limitation increased across soil depths regardless of warming. Write Increased compared to what? And why did it increase?

It is necessary to give a detailed explanation of the formulas used by the authors:

168 EEAC:N = ln(BG+CBH)/ln(NAG+LAP) (1)

EEAC:P = ln(BG+CBH)/ln(AP)

173 Vector length = SQRT [(EEAC:P)2+(EEAC:N)2] (3)

Vector angle = Degrees [ATAN2(EEAC:P, EEAC:N)]

182TERC:N = [(BG+CBH)/(NAG+LAP)]×BC:N/n (5)

TERC:P = [(BG+CBH)/AP]×BC:P/p (6) 183

202-203. Linear mixed-effect 202 modes were used to explore the fixed effects of warming, depth, and their interaction on– Who proposed this approach and what does it mean??

210-212 using linear mixed-effects modes with the block as a random factor. Meanwhile, the 210 SMA regression was used to test the difference between microbial enzymatic stoichiome-211 try and the 1:1 line. Who proposed this approach and what does it mean??

212 Moreover, the relationships between enzymatic stoichiometry and soil 212 environmental, substrate, and microbial properties were examined with Pearson’s corre

What is meant by soil  environmental??, and what is meant by substrate?,

345-347 Our results also revealed that the enzymatic C:N and C:P ratios significantly 346 decreased with soil depth (Figure 2), suggesting that microbial N and P limitation was higher 347 in the subsoil than in the surface soil.

This is an incorrect conclusion; quite the opposite, a decrease in the C:N and C:P ratios suggests an increase in N and P in the deeper layers

311. Changes in microbial resource limitation in the surface soil need add^

the surface soil layer

316-317 In Figure 6, vertical microbial C limitation decreases and microbial nutrient limitation increases along the soil profile, independent of warming. Horizon calculation, warming decreases microbial C limitation and increases microbial nutrient limitation in the surface soil,

The resulting Figure 6 schematically shows that carbon microbial biomass decreases with depth from 0 to 60 cm. but microbial nitrogen and microbial phosphorus, on the contrary, increase. What is very strange is that microbial nitrogen and microbial phosphorus, on the contrary, increase.

In Fig. 6, this is a diagram-schem, and specific values of carbon microbial biomass, microbial nitrogen and microbial phosphorus in the soil profile are not given in the article

And then in Table 6 there is a phrase: Biotic and abiotic factors adjacent to trapezoids 319 are the predominant drivers of the corresponding vertical change in microbial resource limitation. It is not clear why microbial biomass decreases in the soil profile. This phrase should be removed from Figure 6 and explained in detail in the discussion

332 The average enzymatic ratio C:N:P was 1:1.33:1.74 along the soil profile (Figure 2). This is not shown in Figure 2

There are a lot of Abbreviations in the article, they should be in all in the figures, replace the abbreviations with their full meaning.

At the end of the article, there is a transcript of some abbreviations, but the list is incomplete. It needs to be supplemented, for example, there is no F / B, etc.

Figure 5. It is necessary to give % to whole numbers: It is necessary to write 37%, not 37.07%, 63% is needed. 62.93% is not needed

All six figures mainly provide the values of the ratio of various elements and almost no properties of microbial and general soil properties themselves. It is necessary to provide several figures or tables with the absolute amount of carbon, nitrogen and phosphorus, enzymatic activity, the content of fungi and bacteria, etc.

enzymatic ratios C:N and C:P deviated significantly from 1:1, and the vector angle was greater than 45 ° when treated with heating (Figure 2). Note The same can be said about the control soil.

The article can be published after the above-mentioned comments are corrected

With respect 

01/14/2025

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

[Comment 1] The article is devoted to the change in the physicochemical, microbiological and enzymatic characteristics of soils along the profile up to 60 cm in the control soil and in soil with an artificial increase heating by 4 ° C in the tropical forests of China. The article is important and informative, but there are the following comments below.

General comment on the entire text: The authors constantly and frequently use the word limitation throughout the article, and it is often unclear what this word means. The authors should write in the text what they mean by the word limitation. It is better to replace the word limitation in some cases throughout the text with other words, so that it is clearer what is meant - an increase or decrease in the amount of a particular indicator along the profile.

302 For example, 4. Discussion In this study, we provided experimental evidence for the response of soil microbial resource limitation to whole-soil-profile warming and the associated mechanisms in sub-tropical forests (Figure 6). We observed that microbial nutrient (N and P) limitation (replace with content) increased but microbial C limitation decreased consistently along the soil profile regardless of warming. We also found that the vertical variations in microbial N limitation, etc.

[Response] Sorry for the unclear description. To make the description easier to be understand, we first changed the expressions in the revised manuscript, i.e., replacing “microbial resource limitation” by “enzymatic stoichiometry” (Page 9, line 323, 335; Page 11, line 458; Page 12, line 475), “microbial N limitation” by “enzymatic C:N ratio” (Page 9, line 325, 328, 333), “microbial P limitation” by “enzymatic C:P ratio” (Page 9, line 325, 330, 355), “microbial C limitation” by “vector length” (Page 9, line 325, 330, 332; Page 10, line 374; Page 10, line 408). We also added further explanations for microbial C-, N- and P-limitation in the revised manuscript, for example, “soil microbial resource limitation, characterized by enzymatic stoichiometry” (Page 9, line 323; Page 11, line 455; Page 12, line 496), “microbial N limitation, characterized by enzymatic C:N ratio” (Page 10, line 361; Page 11, line 417), “microbial P limitation, characterized by enzymatic C:P ratio” (Page 11, line 443), “microbial C limitation, indicated by vector length” (Page 11, line 441).

 

[Comment 2] Add Title: Warming does not change vertical variations in microbial re-source limitation in subtropical forests at China

[Response] Done as suggested.

 

[Comment 3] In the Abstract: F/B - you need to decipher what this abbreviation means

[Response] We have replaced F/B as ‘fungal-bacterial ratio’ to improve readability of abstract in the revised manuscript (Page 1, line 22, 24, 25, 27; Page 5, line 237, 239…).

 

[Comment 4] In the Abstract: Need Describe the experiment, objects, and methods. Where the object is located, what soil layers and to what depth were studied, what kind of warming was created, etc.

[Response] We have added more descriptions of the warming experiment in the revised manuscript as follows: “Here, we investigated vertical variations (0-60 cm soil layers) in microbial resource limitation and their corresponding responses to warming in subtropical forests in southern China, using a soil warming experiment with heating cables (+ 4 °C) and the enzymatic stoichiometry.” (Page 1, line 14-17).

 

[Comment 5] Need Significantly shorten the captions in all figures. They are often repeated in all figures. and give an explanation of all the abbreviations in all the figures

[Response] We have shorten the captions and given explanations of all the abbreviations in all figures (Page 6, line 261-263; Page 7, line 282-284, line 286-288; Page 8, line 313-316; Page 9, line 319-320; Page 9, line 339-344).

 

[Comment 6] Abstract: 17. microbial nutrient (nitrogen and phosphorus) limitation This it is incorrect to write, because nitrogen and phosphorus are also needed by plants, not just by microbes.

[Response] Sorry for the unclear description. We have rewrote this sentence as follows “Alleviated carbon limitation but aggravated nutrient (nitrogen and phosphorus) limitation for microbial metabolism were observed along soil profiles, regardless of warming treatment.” in the revised manuscript (Page 1, line 17-19).

 

[Comment 7] It is necessary to give a detailed explanation of the formulas used by the authors:

168 EEAC:N = ln(BG+CBH)/ln(NAG+LAP) (1)

EEAC:P = ln(BG+CBH)/ln(AP)

173 Vector length = SQRT [(EEAC:P)2+(EEAC:N)2] (3)

Vector angle = Degrees [ATAN2(EEAC:P, EEAC:N)]

182TERC:N = [(BG+CBH)/(NAG+LAP)]×BC:N/n (5)

TERC:P = [(BG+CBH)/AP]×BC:P/p (6) 183

[Response] Sorry for the unclear description of equations. Microorganisms usually invest in enzyme production to acquire relatively limited elements, thus enzymatic stoichiometry theory can reflect the resource limitation of microbial metabolism. The C-acquisition enzyme usually includes β-1,4-glucosidase (BG) and β-D-cellobiosidase (CBH). The N-acquisition enzyme includes β-1,4-N-acetylglucosaminidase (NAG) and leucine aminopeptidase (LAP). The P-acquisition enzyme refers acid phosphatase (AP).  EEA_C:N and EEA_C:P are the enzymatic ratios of C-, N-, and P-acquisition. Higher EEA_C:N and EEA_C:P mean relatively lower requirements of N and P, indicating lower microbial N and P limitation. Moreover, vector length and angle are the vector analysis of enzymatic stoichiometry. Longer vector length (unitless) indicates greater C requirements of microbial metabolism, that is, higher microbial C limitation. The vector angle < 45° and > 45° denote relatively higher demand of N and P for microorganism, i.e., microbial N- and P-limitation, respectively. Furthermore, TER (TER_C:N and TER_C:P) is the threshold elemental ratio at which microbial metabolic control switches from C limitation to nutrient (N or P) limitation. If the C:N and C:P ratio of resources are greater than the corresponding TER, the lower supply of N and P for microbial metabolism indicates microbial N- and P-limitation, respectively. We have added more description of the equations in the revised manuscript (Page 4, line 171-174, 178-180, 187-193).

 

[Comment 8] 202-203. Linear mixed-effect modes were used to explore the fixed effects of warming, depth, and their interaction on– Who proposed this approach and what does it mean??

210-212 using linear mixed-effects modes with the block as a random factor. Meanwhile, the SMA regression was used to test the difference between microbial enzymatic stoichiometry and the 1:1 line. Who proposed this approach and what does it mean??

[Response] Thanks for the comment. The warming experiment in our study adopted randomized block design. Thus, linear mixed-effects models with block being random factor can efficiently reveal the treatment effects (warming, soil depth and their interaction) on response variables by reducing the influence from differences in blocks [1]. This statistical analysis technique has been applied widely in ecology and soil science [2,3]. We have cited these references in our revised manuscript (Page 5, line 210, 212). Moreover, the type II standard major axis (SMA) regression is a common approach to estimate how one variable scales against another [4]. The most common example of this is allometry, for example, how leaf lifespan scales against leaf mass per area. This method has been widely used in ecology and evolution [5]. We have cited relevant references in the revised manuscript (Page 5, line 217).

 

[Comment 9] 212 Moreover, the relationships between enzymatic stoichiometry and soil environmental, substrate, and microbial properties were examined with Pearson’s correlation.

What is meant by soil environmental??, and what is meant by substrate?

[Response] Soil environmental properties include soil moisture, pH and clay content. Soil substrate properties include soil C:N and C:P ratios. We have added this description in the revised manuscript (Page 5, line 219).

 

[Comment 10] 345-347 Our results also revealed that the enzymatic C:N and C:P ratios significantly decreased with soil depth (Figure 2), suggesting that microbial N and P limitation was higher in the subsoil than in the surface soil.

This is an incorrect conclusion; quite the opposite, a decrease in the C:N and C:P ratios suggests an increase in N and P in the deeper layers.

[Response] Sorry for the unclear description. The Figure 2 showed soil enzyme C:N:P stoichiometry rather than soil C:N:P stoichiometry. The decreased enzymatic C:N and C:P ratios along the soil profile indicate that microorganisms relatively invest more in N- and P-acquiring enzymes, suggesting higher N and P limitation for microbial metabolism in subsoil. We have rewritten this sentence (Page 10, line 371-374) and added explanation of abbreviations for enzymatic stoichiometry in the Figure 2 (Page 7, line 282-284) for readability in the revised manuscript.

 

[Comment 11] 311. Changes in microbial resource limitation in the surface soil need add the surface soil layer

[Response] Done as suggested.

 

[Comment 12] 316-317 In Figure 6, vertical microbial C limitation decreases and microbial nutrient limitation increases along the soil profile, independent of warming. Horizon calculation, warming decreases microbial C limitation and increases microbial nutrient limitation in the surface soil,

The resulting Figure 6 schematically shows that carbon microbial biomass decreases with depth from 0 to 60 cm. but microbial nitrogen and microbial phosphorus, on the contrary, increase. What is very strange is that microbial nitrogen and microbial phosphorus, on the contrary, increase.

In Fig. 6, this is a diagram-schem, and specific values of carbon microbial biomass, microbial nitrogen and microbial phosphorus in the soil profile are not given in the article

And then in Figure 6 there is a phrase: Biotic and abiotic factors adjacent to trapezoids are the predominant drivers of the corresponding vertical change in microbial resource limitation. It is not clear why microbial biomass decreases in the soil profile. This phrase should be removed from Figure 6 and explained in detail in the discussion

[Response] Sorry for the unclear description. The Figure 6 schematically shows the effects of warming and soil depth on microbial resource limitation as indicated by enzymatic stoichiometry rather than that on soil microbial biomass C, N and P along the soil profile. Specifically, the microbial C limitation is indicated by vector length, microbial N limitation by enzymatic C:N ratio, and microbial P limitation by enzymatic C:P ratio. Meanwhile, the biotic and abiotic factors in the Figure 6 are the main drivers of enzymatic stoichiometry. Specifically, under unwarming treatment, the vertical change in vector length and enzymatic C:N and C:P ratios is regulated by soil moisture and fungal-bacterial ratio. Under warming treatment, the vertical change in vector length is regulated by soil moisture and fungal-bacterial ratio, but that in enzymatic C:N and C:P ratios is regulated by soil C:P ratio and fungal-bacterial ratio. To avoid confusion, we have corrected the Figure 6, and replaced the “microbial N limitation” by “enzymatic C:N ratio”, “microbial P limitation” by “enzymatic C:P ratio”, “microbial C limitation” by “vector length” in the revised manuscript (Page 9, line 338). Meanwhile, we rewrote the caption in the revised manuscript as follows: “Conceptual diagram showing effects of warming and soil depth on enzymatic stoichiometry in subtropical forests along the soil profile. Vertically, enzymatic stoichiometry (i.e., vector length and enzymatic C:N and C:P ratios) decrease along the soil profile regardless of warming. Hori-zontally, warming decreases enzymatic stoichiometry in topsoil but has nonsignificant effects in subsoil. Biotic and abiotic factors adjacent to trapezoids are the predominant drivers for vertical changes in enzymatic stoichiometry. F/B, fungal-bacterial ratio; DOC/LP, soil C:P ratio.” (Page 9, line 339-344).

 

[Comment 13] 332 The average enzymatic ratio C:N:P was 1:1.33:1.74 along the soil profile (Figure 2). This is not shown in Figure 2

[Response] Sorry for the incorrect citation of figures. The average enzymatic ratio C:N:P of 1:1.33:1.74 was the arithmetic mean of enzymatic ratio C:N:P along the soil profile. We have rewrote this sentence in the revised manuscript (Page 9, line 346).

 

[Comment 14] There are a lot of Abbreviations in the article, they should be in all in the figures, replace the abbreviations with their full meaning.

At the end of the article, there is a transcript of some abbreviations, but the list is incomplete. It needs to be supplemented, for example, there is no F / B, etc.

[Response] Sorry for the puzzling abbreviations in the article. We have replace the abbreviations with their full meaning in the revised manuscript. Moreover, we have supplemented the transcript of abbreviations in the revised manuscript (Page 13, line 529).

 

[Comment 15] Figure 5. It is necessary to give % to whole numbers: It is necessary to write 37%, not 37.07%, 63% is needed. 62.93% is not needed

[Response] Done as suggested.

 

[Comment 16] All six figures mainly provide the values of the ratio of various elements and almost no properties of microbial and general soil properties themselves. It is necessary to provide several figures or tables with the absolute amount of carbon, nitrogen and phosphorus, enzymatic activity, the content of fungi and bacteria, etc.

[Response] Thanks for comments. We agree with the reviewer that soil microbial and physiochemical properties are essential in this study. However, our study concentrates on the relative resource limitation of microbial metabolism, which is mainly regulated by soil environmental properties, microbial biomass stoichiometry and soil resource stoichiometry. To reveal the drivers of microbial resource limitation, we tend to provide these important factors in the main figures. Moreover, we have provided the values of soil microbial and physiochemical properties in the supplementary figures, such as soil dissolved C and N, available P, enzyme activity, microbial biomass C, N and P, and bacterial and fungal abundance in the revised manuscript (Page 14, line 540). Thanks for your understanding!

 

[Comment 17] enzymatic ratios C:N and C:P deviated significantly from 1:1, and the vector angle was greater than 45 ° when treated with heating (Figure 2). Note The same can be said about the control soil.

[Response] Thanks for the comment. To make the description more concise, we have rewrote this sentence as follow: “Our results showed that enzymatic C:N and C:P ratios and vector angle under warming treatment exhibited similar vertical pattern to those under the control treatment (Figure 2)” in the revised manuscript (Page 10, line 405-406).

 

[Comment 18] The English could be improved to more clearly express the research.

[Response] Thanks for the comment. We have asked a native speaker for editing English, and we hope that the language is satisfactory and become acceptable for publication.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled 'Warming does not change vertical variations in microbial resource limitation in subtropical forests' aims to investigate the effects of warming on microbial resource limitation across soil profiles in subtropical forests. This manuscript addresses the crucial topic of the impact of warming on microbial resource limitation across soil profiles in subtropical forests, which is strong in its experimental design and presentation of results. However, some areas require clarification, particularly the discussion of the results.

 

L143, L176 - Citation formatting for reference is inconsistent.

L180-187 Enzymatic stoichiometry relationships - the dimensionless normalization constants n and p (C:N and C:P) are described as 2.718^a and 2.718^b, with a and b being intercepts derived from SMA regressions. I suggest to verify the calculations for these constants and clarify how a and b differ across regressions.

 

In the discussion section, the authors emphasize that microbial carbon limitation decreases with depth while nitrogen and phosphorus limitations increase. The mechanistic explanations for these vertical trends, particularly in the context of microbial community composition and soil moisture, need to be expanded. More detailed discussion is needed on the interactions between abiotic and biotic drivers (e.g., fungal-to-bacterial ratios) and how these are modulated by warming.

Furthermore, the manuscript concludes that warming did not significantly affect microbial resource limitation below 20 cm. This observation lacks a strong mechanistic explanation. Could limited warming penetration, microbial thermal acclimation, or substrate availability explain this phenomenon? Including additional discussion or supporting references would strengthen this point.

 

All figure captions should include the abbreviation's meanings.

 

Table A2 includes critical statistical results but is relegated to supplementary materials. Consider integrating Table A2 into the main text. I also suggest to highlight the significant results, for example, in bold.

Author Response

[Comment 1] The manuscript titled 'Warming does not change vertical variations in microbial resource limitation in subtropical forests' aims to investigate the effects of warming on microbial resource limitation across soil profiles in subtropical forests. This manuscript addresses the crucial topic of the impact of warming on microbial resource limitation across soil profiles in subtropical forests, which is strong in its experimental design and presentation of results. However, some areas require clarification, particularly the discussion of the results.

[Response] Thanks for the reviewer’s positive and insightful comments. These comments, combined with those listed below inspired us to have a deeper thinking on this issue, and thus guided us to conduct a thorough revision of the original manuscript. To address the reviewer’s comments listed below, we made the following major changes in the revised manuscript. First, we have revised the method description to avoid confusion in the Materials and Methods section (Page 4, line 188-190). Second, we have added further mechanistic explanations for the vertical changes in soil moisture, soil C:P ratio and fungal-bacterial ratio, as well as the nonsignificant response of enzymatic stoichiometry below 20 cm soil depth in the Discussion section (Page 10, line 381-382, 385-388; Page 11, line 419-424, line 435-437; Page 12, line 479-492). Third, we have emphasized statistical results of soil properties and enzymatic stoichiometry in tables and given explanations for all abbreviations in all figures (Page 6, line 255). Fourth, we have checked and corrected the citation formatting for reference throughout the manuscript. After incorporating these comments, we feel that the revised manuscript has been significantly improved. Thank you!

 

[Comment 2] L143, L176 - Citation formatting for reference is inconsistent.

[Response] Sorry for the incorrect citation formatting of references. We have also carefully checked throughout the manuscript, revised the incorrect citation formatting.

 

[Comment 3] L180-187 Enzymatic stoichiometry relationships - the dimensionless normalization constants n and p (C:N and C:P) are described as 2.718^a and 2.718^b, with a and b being intercepts derived from SMA regressions. I suggest to verify the calculations for these constants and clarify how a and b differ across regressions.

[Response] Thanks for the comments. The a is the intercept of a type II standard major axis (SMA) regression for C-acquisition enzymes versus N-acquisition enzymes (intercept = -3.08, slope = 1.54). Similarly, the b is the intercept of a type II standard major axis (SMA) regression for C-acquisition enzymes versus P-acquisition enzymes (intercept = 0.03, slope = 0.62). Thus, the values of a and b are two independent constants and may be not be compared together. To make the description more clear, we have redescribe the constants in the revised manuscript as follows: “The a is the intercept of a type II standard major axis (SMA) regression for ln(BG+CBH) versus ln(NAG+LAP). The b is the intercept of SMA regression for ln(BG+CBH) versus ln(AP).”(Page 4, line 188-190).

 

[Comment 4] In the discussion section, the authors emphasize that microbial carbon limitation decreases with depth while nitrogen and phosphorus limitations increase. The mechanistic explanations for these vertical trends, particularly in the context of microbial community composition and soil moisture, need to be expanded. More detailed discussion is needed on the interactions between abiotic and biotic drivers (e.g., fungal-to-bacterial ratios) and how these are modulated by warming.

Furthermore, the manuscript concludes that warming did not significantly affect microbial resource limitation below 20 cm. This observation lacks a strong mechanistic explanation. Could limited warming penetration, microbial thermal acclimation, or substrate availability explain this phenomenon? Including additional discussion or supporting references would strengthen this point.

[Response] Thanks for the comments. We have added mechanistic explanations for the vertical changes in biotic and abiotic factors and their response in the revised manuscript to warming as follows: “The relatively abundant soil water reserves in deep soil layers might be due to the vertical migration of precipitation along the soil profiles [63].” (Page 10, line 381-382); “Meanwhile, the relatively higher fungal-bacterial ratio in deep soil layers might be linked to the recalcitrant substrates, supported by the higher ratio of mineral-associated organic C to particulate organic C in subsoil in subtropical forests [67].” (Page 10, line 385-388); “Warming usually has greater stimulative effects on water evapotranspiration in topsoil than in subsoil [25]. The remarkable reduction in soil moisture in surface soil layer may increase fungal dominance, as fungi show greater resistance and resilience to envi-ronmental change than bacteria [71]. This view was confirmed by the decreased soil moisture and increased fungal-bacterial ratio in topsoil in our study (Figure 1), leading to the increasing pattern of soil moisture and fungal-bacterial ratio along soil profiles.” (Page 11, line 419-424); “The vertical transport of dissolved organic matter and deep roots, as described above, may lead to a relatively higher soil C:P ratio in deep soil layers [63].” (Page 11, line 435-437). We have also added mechanistic explanations for nonsignificant response of enzymatic stoichiometry below 20 cm soil depth in the revised manuscript to warming as follows: “Such a contradiction might be ascribed to the nonsignificant response of soil physio-chemical and microbial properties to warming in deep soil profiles. Warming usually decreases soil water content and stimulates the decomposition of soil organic matter, which may trigger negative or positive effects on microbial metabolism [80,81]. How-ever, no significant effects of warming on soil moisture and substrate supply, such as dissolved organic C, total dissolved N, and available P, were observed in deep soil in our study (Figure A3). Such a phenomenon might be caused by the limited water evapotranspiration and microbial activity in deep soil layers [82]. Furthermore, a sim-ilar pattern of microbial biomass and community structure (indicated by fun-gal-bacterial ratio) was also observed in the subsoil (Figure A3). Such a phenomenon might be linked to microbial thermal acclimation, which had been observed in other whole-soil-profile warming studies [26]. The constant soil moisture, substrate supply and microbial community might cause no significant response of microbial nutrient limitation to warming.” (Page 12, line 479-492).

 

[Comment 5] All figure captions should include the abbreviation's meanings.

[Response] We have given an explanation of all abbreviations in all figures in the revised manuscript (Page 6, line 261-263; Page 7, line 282-284, line 286-288; Page 8, line 313-316; Page 9, line 319-320; Page 9, line 339-344).

 

[Comment 6] Table A2 includes critical statistical results but is relegated to supplementary materials. Consider integrating Table A2 into the main text. I also suggest to highlight the significant results, for example, in bold.

[Response] Thanks for the comments. We have integrated the statistical results of soil properties and enzymatic stoichiometry in the main text, and significant results are highlighted in bold in the revised manuscript (Page 6, line 255).

 

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3. Zhang, Q.; Qin, W.; Feng, J.; Li, X.; Zhang, Z.; He, J.-S.; Schimel, J.P.; Zhu, B. Whole-soil-profile warming does not change microbial carbon use efficiency in surface and deep soils. Proc. Natl. Acad. Sci. USA 2023, 120, e2302190120.
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Author Response File: Author Response.pdf