Review Reports - Calcium-Associated Cellular Integrity and MAPK/cAMP Signaling Are Associated with Low-Temperature Acclimation in <i>Schizophyllum commune</i> 20R-7-F01

Round 1

Reviewer 1 Report

The authors are to be commended for obtaining interesting and insightful results. The manuscript is well structured and addresses a topic of considerable relevance to both mycology and biotechnology. A major strength of the study is the understanding the role of physiological and molecular responses of Schizophyllum commune to low-temperature stress. The work expands how low-temperature may affect cell wall remodeling, antioxidant responses, and MAPK, cAMP, and Ca²⁺ signaling. This research topic is important because it contributes to a better understanding of environmental adaptation in basidiomycete fungi.

Overall, the topic is of practical relevance, and the manuscript cites an body of literature, reflecting the major research trajectories and recent developments in this field. The manuscript is clearly structured and the material is presented in a logical and consistent way. The introduction clearly and scientifically demonstrates the relevance and feasibility of the study. The experimental design is appropriately planned. Research methodology is presented clearly and understandably. A significant amount of material has been obtained by the authors, which is well presented and graphically illustrated. Obtained material is easy to understand and comprehend. The concluding remarks are justified and summarised the work carried out. The article is of good quality and is recommended for publication.

I have only one comment. The scale bar is not visible in the Figute 7B. Please add a clearly visible scale bar.

Author Response

Comments 1: I have only one comment. The scale bar is not visible in the Figute 7B. Please add a clearly visible scale bar. Response 1: Thank you for pointing this out. We sincerely apologize for this oversight. We have revised Figure 7 to ensure the scale bar is clearly visible within the image. We have also modified the figure legend for Figure 7 to distinguish between the 10 μm scale bar for optical microscopy and the 500 nm scale bar for transmission electron microscopy (TEM). Furthermore, we have corrected the description of Figure 7B to clarify that Evans blue staining was used to assess membrane integrity rather than cell wall integrity. Line: 483-499.

Reviewer 2 Report

This manuscript addresses a relevant and interesting question: how the model basidiomycete Schizophyllum commune responds to low-temperature stress. The authors have assembled a broad dataset covering growth, development, cell wall composition, oxidative stress, and three signaling pathways. The topic fits well within the scope of Journal of Fungi, and the work has clear potential to contribute to our understanding of fungal cold adaptation.

However, the manuscript currently has several conceptual, methodological, and reporting issues that must be addressed before it can be considered for publication. Many of these appear to stem from study design and data presentation choices rather than from fundamental flaws, and most are straightforward to fix. I have organized my comments into (A) major concerns requiring new data or reanalysis, and (B) reporting and presentation issues that can be resolved without new experiments. I hope these comments are helpful and constructive.

Major Concerns

1. Single-isolate design and unacknowledged strain-specificity

The study uses a single S. commune isolate (20R-7-F01, CGMCC 5.2202), originally isolated from deep subseafloor coal-bearing sediments (IODP Expedition 337). However:

The Introduction frames S. commune as a cosmopolitan, temperate/cool-habitat wood-decayer.
The Abstract and Conclusions generalize the findings to "S. commune" as a species.
The deep-sea origin of the strain is never discussed as a potential confound.
There is no justification for using a single isolate, and no acknowledgment that intraspecific variation in cold tolerance is unexamined.

How to fix:

State explicitly in the Methods why this strain was chosen (e.g., it is the lab's established model, genome available, prior work).
Reframe the study as strain-specific throughout the title, abstract, and conclusions (e.g., "S. commune strain 20R-7-F01").
Add a Limitations paragraph in the Discussion addressing:
- The deep-sea origin of the strain and how it may differ from temperate isolates.
- The lack of intraspecific comparison.
- That the findings are hypothesis-generating, not species-level conclusions.

2. Lack of mechanistic evidence for pathway involvement (MAPK, cAMP, Ca²⁺)

The manuscript claims that MAPK, cAMP, and Ca²⁺ signaling are involved in cold adaptation, but the evidence is largely correlative:

U0126 treatment reduces biomass and downregulates MAPK genes at 10 °C. However, there is no 30 °C + U0126 control, so it is unclear whether the effect is cold-specific or simply general growth inhibition. There is also no verification that U0126 actually inhibits MAPK phosphorylation (e.g., by Western blot against phospho-MAPK).
Only transcript levels of five genes are reported (Figure 6). There is no 30 °C control for these genes, no measurement of intracellular cAMP, and no PKA activity assay. The claim that "cAMP signaling is activated" is not supported.
Intracellular Ca²⁺ is measured, and EDTA chelation impairs cell wall integrity (Evans blue). However, EDTA is not Ca²⁺-specific — it chelates Mg²⁺, Fe²⁺, Zn²⁺, and other divalent cations essential for cell wall integrity. No Ca²⁺ rescue or specific Ca²⁺ manipulation (e.g., BAPTA-AM, Ca²⁺ channel blockers) is included.

How to fix:

Add a 30 °C + U0126 control. If possible, verify MAPK inhibition by Western blot (phospho-MAPK vs. total MAPK).
Add a 30 °C control for all cAMP genes (Figure 6). If possible, measure intracellular cAMP levels and/or PKA activity at 10 °C vs. 30 °C.
Replace or supplement EDTA with a Ca²⁺-specific chelator (BAPTA-AM) or a Ca²⁺ channel blocker. Include a Ca²⁺ rescue (EDTA + excess Ca²⁺) to show specificity.

3. Incomplete and unclear Methods

Several methodological details are missing or unclear, which prevents reproducibility:

Fruiting body culture: The authors state cultures were grown at 30 °C for 168 h, then exposed to "600 h light treatment (12 h day/12 h night)." It is unclear:
- What temperature was used during the 600 h light treatment?
- How low-temperature conditions were applied to fruiting bodies.
- How the timeline in Figure 2A was recorded (e.g., time to primordia, fruiting body, maturation).
Basidiospore germination: The method for quantifying germination is vague. Was it spore length, germination percentage, or both? How many spores were counted? What was the criterion for "germinated"?
Evans blue staining: The protocol appears in section 2.6, but the Results (3.7) describe different conditions (10 °C ± EDTA). How was EDTA applied (concentration, duration)? Was it added to the culture medium or to the staining solution?
Chitin content: The unit "mg kg⁻¹" is unusual. Is this per dry weight or wet weight? How was the mycelium harvested and processed?
Liquid MM composition: Not described; refer to prior work or provide details.

How to fix:

Provide a detailed, step-by-step Methods section for each experiment, including temperature, duration, replicate number, and how measurements were taken.
For fruiting body and germination experiments, include a timeline figure or supplementary table showing exactly when each developmental stage was scored and at what temperature.
Specify EDTA concentration and application method clearly.

4. Overinterpretation of correlative data

The Abstract and Conclusions state that S. commune adapts to low temperature through "coordinated regulation of calcium signaling, cell wall remodeling, antioxidant defense, and MAPK/cAMP signaling pathways." This is stronger than the data support. The data show changes in these pathways, not coordinated regulation or causality.

How to fix:

Soften the language throughout. Replace "demonstrates" with "suggests," "coordinates" with "is associated with," and "essential for" with "may contribute to."
In the Discussion, explicitly state that the study is descriptive and hypothesis-generating, and that causal testing (genetic manipulation, biochemical assays) is needed.
Add a Limitations paragraph that acknowledges:
- Single isolate.
- Correlative nature of pathway data.
- Lack of direct cAMP/PKA measurements.
- Non-specific Ca²⁺ chelation.
- Lack of proteomic/metabolomic data.

The reference list is incomplete. References 47–52 are cited in the Discussion but do not appear in the list. Reference 10 is dated 2026, which may be a placeholder. "Zhang et al., 2021" is cited in the Introduction but not listed.
There are numerous typographical errors, e.g., "basisipore" → "basidiospore" and "GSG" → "GSSG".
Most experiments use n = 3, but Figure 1 uses n = 20. This discrepancy should be explained.
Also, the error bars appear inconsistent with n = 20 — for example, the 5 °C bar has a very large error bar, while the 30 °C bar has a very small one. If n = 20 biological replicates, the SEM should be much smaller.

Author Response

Comments 1:

Single-isolate design and unacknowledged strain-specificity

The study uses a single S. commune isolate (20R-7-F01, CGMCC 5.2202), originally isolated from deep subseafloor coal-bearing sediments (IODP Expedition 337). However:

The Introduction frames S. commune as a cosmopolitan, temperate/cool-habitat wood-decayer.
The Abstract and Conclusions generalize the findings to "S. commune" as a species.
The deep-sea origin of the strain is never discussed as a potential confound.
There is no justification for using a single isolate, and no acknowledgment that intraspecific variation in cold tolerance is unexamined.

How to fix:

State explicitly in the Methods why this strain was chosen (e.g., it is the lab's established model, genome available, prior work).
Reframe the study as strain-specific throughout the title, abstract, and conclusions (e.g., "S. commune strain 20R-7-F01").
Add a Limitations paragraph in the Discussion addressing:

The deep-sea origin of the strain and how it may differ from temperate isolates.
The lack of intraspecific comparison.
That the findings are hypothesis-generating, not species-level conclusions.

Response 1:

We agree with this comment. This content has been added to the revised "Methods" section. The added content is: "This strain was selected because it is an established model strain in our laboratory and has been widely used in our previous research; furthermore, its well-defined genetic background and comprehensive genomic information make it highly suitable for this study.".
We agree that the findings of this study should be appropriately presented as strain-specific rather than generalized to the species as a whole. Accordingly, we have revised the title, abstract, and conclusions to explicitly refer to S. commune 20R-7-F01. In addition, we carefully reviewed the entire manuscript, including the Introduction, Results, Discussion, and figure legends, and revised relevant statements to avoid overgeneralization to S. commune as a species. These revisions ensure that the results and conclusions are clearly framed as specific to S. commune 20R-7-F01.
We acknowledge that certain limitations should be clearly stated when interpreting the research findings. Therefore, we have added a new paragraph at the end of the "Discussion" section outlining the following points: (i) the strain originated from the deep sea, and its physiological and/or ecological characteristics may differ from those of isolates from other environments; (ii) the lack of intraspecific comparisons; and (iii) the need to view the study results as the proposal of a hypothesis rather than definitive conclusions at the species level. The specific details are as follows: " Several limitations should be considered when interpreting the results of this study. First, the strain examined was isolated from a deep-sea environment; its physiological, metabolic, and ecological characteristics may differ from those of isolates originating from other environments. Different fungal strains exhibit varying capacities to adapt to low-temperatures; consequently, the physiological responses and underlying mechanisms observed in this specific strain may not necessarily be representative of strains from other environments. Furthermore, fungal responses to temperature depend on the species and developmental stage; for certain fungi that produce large fruiting bodies, a drop in temperature under conditions of adequate moisture, light, and ventilation may actually promote rather than delay specific developmental transitions. Therefore, the responses observed in the S. commune 20R-7-F01 should not be generalized to all basidiomycetes. Second, the study did not include multiple isolates of the same species, which limited the ability to assess intraspecific variation and distinguish between strain-specific and species-level characteristics. Third, this study is descriptive and hypothesis-generating; the pathway-related data reflect correlations rather than causal relationships, and causal validation through gene manipulation and biochemical analyses is still required. Fourth, the lack of proteomic and metabolomic data limits a comprehensive understanding of the downstream molecular events. Finally, the findings should be viewed as preliminary results intended to generate hypotheses rather than as definitive conclusions at the species level. Future comparative studies involving multiple strains from diverse geographical regions and environments will be conducted to determine the degree to which the observed characteristics are conserved within the species.".

Comments 2:

Lack of mechanistic evidence for pathway involvement (MAPK, cAMP, Ca²⁺)

The manuscript claims that MAPK, cAMP, and Ca²⁺ signaling are involved in cold adaptation, but the evidence is largely correlative:

U0126 treatment reduces biomass and downregulates MAPK genes at 10°C. However, there is no 30°C + U0126 control, so it is unclear whether the effect is cold-specific or simply general growth inhibition. There is also no verification that U0126 actually inhibits MAPK phosphorylation (e.g., by Western blot against phospho-MAPK).
Only transcript levels of five genes are reported (Figure 6). There is no 30°C control for these genes, no measurement of intracellular cAMP, and no PKA activity assay. The claim that "cAMP signaling is activated" is not supported.
Intracellular Ca²⁺ is measured, and EDTA chelation impairs cell wall integrity (Evans blue). However, EDTA is not Ca²⁺-specific — it chelates Mg²⁺, Fe²⁺, Zn²⁺, and other divalent cations essential for cell wall integrity. No Ca²⁺ rescue or specific Ca²⁺ manipulation (e.g., BAPTA-AM, Ca²⁺ channel blockers) is included.

How to fix:

Add a 30°C + U0126 control. If possible, verify MAPK inhibition by Western blot (phospho-MAPK vs. total MAPK).
Add a 30°C control for all cAMP genes (Figure 6). If possible, measure intracellular cAMP levels and/or PKA activity at 10°C vs. 30°C.
Replace or supplement EDTA with a Ca²⁺-specific chelator (BAPTA-AM) or a Ca²⁺ channel blocker. Include a Ca²⁺ rescue (EDTA + excess Ca²⁺) to show specificity.

Response 2:

We agree with this comment. The addition of the 30°C treatment helps to further demonstrate of U0126 action under low-temperature conditions. In fact, we had already completed this experiment in our preliminary work, but the data were not included in the initial draft. These data have now been added to Figure 5 and briefly described in the main text. The revised description reads: "Biomass analysis showed that the mycelial biomass of the U0126 treated group was significantly lower than that of the untreated control group (Figure 5A), indicating that inhibition of the MAPK signaling pathway significantly inhibits the vegetative growth of S. commune 20R-7-F01 under low-temperature stress. In addition, the biomass of the U0126 treated group at 30 °C was also lower than that of the untreated group.".

The gene expression data have been added to Figure 5B, and the following description has been added: "In contrast, at 30 °C, the relative expression levels of all genes were generally low, yet U0126 treatment still significantly downregulated the expression of all genes examined. (Figure 5B).".

We understand the importance of validating MAPK inhibition via Western blot. However, after careful consideration, we have not included this experiment in the current revision for the following reasons:

1) The core conclusions of this study do not depend on the quantification of MAPK inhibition. The main conclusions are based on the changes in biomass and the significant downregulation of MAPK pathway gene transcription after U0126 treatment, which constitute evidence at the functional phenotypic and transcriptional levels. Western blot verifies protein phosphorylation levels, which represents mechanistic evidence at a different level and is not a prerequisite for supporting the core conclusions of this study.

2) Under low-temperature conditions, phosphorylated MAPK signals are weak, and the sensitivity and quantitative reliability of Western blot are limited. At 10°C, fungal metabolism and signaling pathway activity are generally reduced, and the basal phosphorylated MAPK signal itself is weak. On this basis, further detecting the inhibitory effect of U0126 is prone to problems such as signals approaching the detection limit, large deviations in band quantification, and poor reproducibility, making it difficult to provide quantitative data that meet statistical requirements.

3) Antibody, sample, and independent replicate conditions were limited within the revision period. Obtaining reliable quantitative results for phospho-MAPK and total MAPK requires specific antibodies, sufficient protein amounts, and at least three independent biological replicates. Under low-temperature slow-growth conditions, mycelial biomass accumulation is limited, making it difficult to complete condition optimization and stable replication within the revision period.

4) To avoid including data with insufficient evidence strength. We believe that supplementing Western blot data without ensuring quantitative reliability and reproducibility could instead introduce misleading results.

Nevertheless, the reviewer's suggestion has provided us with a valuable direction for future research, and we will include the molecular verification of MAPK inhibition as a future research direction.

We agree with this comment. In the previous version, we designated 30°C as the control condition but did not include the corresponding data in the figures; the relevant data have now been added and are presented in Figure 6A. Additionally, intracellular cAMP levels were measured at 10°C and 30°C, and the results are shown in Figure 6B. Figure 6 has been redrawn. A description of Figure 6B has also been added to the revised version: "At the same time, we measured the cAMP content in mycelial samples. As shown in Figure 6B, the cAMP content in the 10 °C treatment group (0.17 μg g⁻¹ FW) was significantly higher than that in the 30 °C treatment group (0.12 μg g⁻¹ FW), indicating that low-temperature significantly promotes cAMP accumulation.Together, these transcriptional changes and elevated cAMP levels suggest that cAMP-associated responses may participate in the low-temperature response of S. commune 20R-7-F01.".

The determination of cAMP content has also been supplemented in the Materials and Methods section. Line: 265-275.

We agree with this comment. In fact, we had considered this issue during the initial stages of our experimental design and conducted experiments using BAPTA-AM treatment alone. As shown in the revised Figure 7B, the results for the BAPTA-AM-treated group were consistent with those of the EDTA-treated group; both groups exhibited significant Evans blue staining under low-temperature conditions. We also seriously considered performing a Ca²⁺ rescue experiment (EDTA + excess Ca²⁺). However, we were concerned that adding excess Ca²⁺ to the culture system might not only alter the Ca²⁺ concentration but also disrupt the balance of other ions, thereby introducing confounding factors that would complicate the interpretation of the results. Therefore, we opted for BAPTA-AM-mediated specific chelation as an alternative approach to directly verify the specific role of Ca²⁺ in maintaining cell membrane integrity (Figure 7B and 7C). Furthermore, we examined cell wall integrity using transmission electron microscopy (TEM), which further confirmed the critical role of Ca²⁺ in maintaining cell wall integrity (Figure 7D). We appreciate this constructive suggestion from the reviewer, as it has significantly strengthened the rigor of our experimental design and the reliability of our conclusions. Lines: 465-479.

Comments 3:

Incomplete and unclear Methods

Several methodological details are missing or unclear, which prevents reproducibility:

Fruiting body culture: The authors state cultures were grown at 30 °C for 168 h, then exposed to "600 h light treatment (12 h day/12 h night)." It is unclear:

What temperature was used during the 600 h light treatment?
How low-temperature conditions were applied to fruiting bodies.
How the timeline in Figure 2A was recorded (e.g., time to primordia, fruiting body, maturation).

Basidiospore germination: The method for quantifying germination is vague. Was it spore length, germination percentage, or both? How many spores were counted? What was the criterion for "germinated"?
Evans blue staining: The protocol appears in section 2.6, but the Results (3.7) describe different conditions (10 °C ± EDTA). How was EDTA applied (concentration, duration)? Was it added to the culture medium or to the staining solution?
Chitin content: The unit "mg kg⁻¹" is unusual. Is this per dry weight or wet weight? How was the mycelium harvested and processed?
Liquid MM composition: Not described; refer to prior work or provide details.

How to fix:

Provide a detailed, step-by-step Methods section for each experiment, including temperature, duration, replicate number, and how measurements were taken.
For fruiting body and germination experiments, include a timeline figure or supplementary table showing exactly when each developmental stage was scored and at what temperature.
Specify EDTA concentration and application method clearly.

Response 3:

We agree with this comment. We have revised the entire text.
We agree with this comment. Relevant content has been added to the section on fruiting body cultivation; the description now reads: "Fruiting body culture: To assess fruiting body development and gene expression associated with fruiting body formation, mycelial blocks with a diameter of 2 mm were cultured on solid MM medium at 10 °C and 30 °C for 168 h. The cultures were maintained at their respective temperatures and then exposed to a 600 h light treatment (12 h day/12 h night). Throughout the entire cultivation process, all cultures were kept in an artificial climate chamber to ensure stable temperature and light conditions.".

Relevant content has been added to the section on basidiospore germination; the description now reads: "Basidiospore germination: Collected basidiospores (on the fruiting bodies of S. commune 20R-7-F01) were suspended in sterile deionized water at a concentration of 1 × 10⁸mL^-1. Then, 1 mL of the basidiospore suspension was transferred to a culture flask containing 15 mL of liquid MM medium (the basidiospore concentration at this point was 10⁶mL^-1) [33]. The suspension was incubated at 10 or 30 °C for a specified time, and the basidiospore length was measured. The germination process of basidiospores involves several stages: activation, isotropic growth, and polarized growth. Initially, basidiospores undergo activation (during which its length remains virtually unchanged), triggering the germination process. This is followed by isotropic growth, which is marked by swelling and water uptake, along with wall growth (during which the basidiospore length increases slowly). Polarized growth begins once wall deposition becomes polarized, forming a germ tube (at which point the basidiospore length increases significantly) [38]. In this study, a basidiospore was considered germinated when a visible germ tube emerged, marking the onset of polarized growth. During the germination of basidiospores, observations were made at 2 h intervals; at each time point, 20 basidiospores were randomly examined under a light microscope, and the length of each individual basidiospore was measured.".

At the same time, we have redrawn Figure 2. The time course is already shown in the figure. Line: 348.

We agree with this comment. The relevant information has been added to the Materials and Methods section: "Subsequently, 1 g (wet weight) of inoculum was transferred into a 250 mL Erlenmeyer flask containing 150 mL of MM, either with or without the addition of 10 μM U0126 [35] or 10 mM EDTA (Ca²⁺ chelating agent) [36] or 20 μM BAPTA-AM (a membrane-permeable, Ca²⁺ specific chelator) [37]. U0126, a MEK inhibitor commonly used to target ERK-type MAPK signaling in other systems, was used as a pharmacological treatment in this study. Its molecular target and the effect on mitogen-activated protein kinase (MAPK) phosphorylation were not directly assessed in S. commune 20R-7-F01. EDTA was directly dissolved in the liquid MM to a final concentration of 10 mM, while BAPTA-AM was first dissolved in DMSO as a stock solution and then added to the liquid MM to a final concentration of 2 mM (with a final DMSO concentration of ≤ 0.1%, v/v). An equivalent volume of DMSO was added to the control group to exclude solvent effects. The dissolution method for U0126 is the same as that for BAPTA-AM.".

At the same time, we have revised the following:

1) The records regarding the time required for primordium initiation, fruiting body formation, and maturation; the description in the Materials and Methods section now reads: "After the mycelium shifts from vegetative to reproductive growth, the small, dark brown protrusions visible to the naked eye on the surface of the substrate are defined as primordia; the time of their initial appearance serves as the recording point. Further differentiation of the primordia into young fruiting bodies characterized by distinct stipe and cap structures marks the stage of fruiting body formation; the time when the differentiation of the cap and stipe is complete and the fruiting body's outline is clearly discernible is used as the recording point for this stage. Fruiting body maturity is reached when the fruiting body develops fully, the cap expands completely with flat edges, and spore release has either not yet begun or is just commencing; the time of complete cap expansion serves as the recording point for this stage. Observations are conducted every 6 h following the start of the experiment, with detailed records kept of the time required to reach each developmental stage.".

2) The unit for chitin content has been revised: μg g^-1 FW represents the chitin content per g of fresh weight (Line: 373-374). The harvesting and processing of mycelium are now described as follows: The cultured mycelia were washed three times with sterile deionized water to remove residual culture medium components. Subsequently, the collected mycelia were blotted dry with sterile absorbent paper, and an accurately weighed 0.1 g sample was placed in 6 mL of 0.5 mol L^-1 hydrochloric acid solution for hydrolysis (110 °C, 2 h). (Line: 200-204).

3) Liquid MM components have been supplemented; see details below: "Detailed information about the habitat, minimal medium (MM, 20 g C₆H₁₂O₆, 1.5 g C₄H₈N₂O₃, 1.0 g K₂HPO₄, 0.46 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 0.12 mg Vitamin B1, 5 mg FeC1₃·6H₂O, and trace elements in 1 L of deionized water), and culture conditions for this strain (dikaryotic strain) has been previously described [32, 33].". Line: 111-115.

Comments 4:

Overinterpretation of correlative data

How to fix:

Soften the language throughout. Replace "demonstrates" with "suggests," "coordinates" with "is associated with," and "essential for" with "may contribute to."
In the Discussion, explicitly state that the study is descriptive and hypothesis-generating, and that causal testing (genetic manipulation, biochemical assays) is needed.
Add a Limitations paragraph that acknowledges:

Single isolate.
Correlative nature of pathway data.
Lack of direct cAMP/PKA measurements.
Non-specific Ca²⁺ chelation.
Lack of proteomic/metabolomic data.

Response 4:

We agree with this comment. Soften the language throughout the text has been revised.
We agree with this comment. Supplementary notes on the parts already discussed: Third, this study is descriptive and hypothesis-generating; the pathway-related data reflect correlations rather than causal relationships, and causal validation through gene manipulation and biochemical analyses is still required. Line: 606-608.
We agree with this comment. Expanded the discussion on the points already addressed. Data on the direct measurement of cAMP/PKA and data related to specific Ca²⁺ chelators have been added.

The discussion regarding the additional limitations is as follows: Several limitations should be considered when interpreting the results of this study. First, the strain examined was isolated from a deep-sea environment; its physiological, metabolic, and ecological characteristics may differ from those of isolates originating from other environments. Different fungal strains exhibit varying capacities to adapt to low-temperatures; consequently, the physiological responses and underlying mechanisms observed in this specific strain may not necessarily be representative of strains from other environments. Furthermore, fungal responses to temperature depend on the species and developmental stage; for certain fungi that produce large fruiting bodies, a drop in temperature under conditions of adequate moisture, light, and ventilation may actually promote rather than delay specific developmental transitions. Therefore, the responses observed in the S. commune 20R-7-F01 should not be generalized to all basidiomycetes. Second, the study did not include multiple isolates of the same species, which limited the ability to assess intraspecific variation and distinguish between strain-specific and species-level characteristics. Third, this study is descriptive and hypothesis-generating; the pathway-related data reflect correlations rather than causal relationships, and causal validation through gene manipulation and biochemical analyses is still required. Fourth, the lack of proteomic and metabolomic data limits a comprehensive understanding of the downstream molecular events. Finally, the findings should be viewed as preliminary results intended to generate hypotheses rather than as definitive conclusions at the species level. Future comparative studies involving multiple strains from diverse geographical regions and environments will be conducted to determine the degree to which the observed characteristics are conserved within the species.

Section 3.6 includes additional data on the direct measurement of cAMP. Section 3.7 includes additional results from experiments involving the specific chelation of Ca²⁺.

Comments 5:

The reference list is incomplete. References 47–52 are cited in the Discussion but do not appear in the list. Reference 10 is dated 2026, which may be a placeholder. "Zhang et al., 2021" is cited in the Introduction but not listed.

Response 5:

We agree with this comment. We rechecked the reference list and all in-text citations. The previously missing references and the numerical citation for the former “Zhang et al., 2021” citation have been corrected.

Comments 6:

There are numerous typographical errors, e.g., "basisipore" → "basidiospore" and "GSG" → "GSSG".

Response 6:

We have now conducted a full language and formatting revision of the manuscript and corrected typographical errors, inconsistent abbreviations, repeated text in the RT-qPCR section, gene/term formatting, units, temperature spacing, figure-legend wording, and bibliographic details.

Comments 7:

Most experiments use n = 3, but Figure 1 uses n = 20. This discrepancy should be explained.

Response 7:

Figure 1 involves the measurement of mycelial growth rates and biomass accumulation. To ensure the representativeness and statistical power of the results, we used a sample size of n = 20 replicates per group. A larger sample size helps to more accurately reflect true trends at the population level and minimizes the confounding effects of individual variation on the conclusions.

Comments 8:

Also, the error bars appear inconsistent with n = 20 — for example, the 5 °C bar has a very large error bar, while the 30 °C bar has a very small one. If n = 20 biological replicates, the SEM should be much smaller.

Response 8:

We rechecked the Figure 1 source data and redrew the figure using the correct mean ± S.E. values for the 20 independent biological replicates. We have retained the explicit sample size in the legend and verified that the displayed error bars correspond to the recalculated values.

Reviewer 3 Report

Dear Authors,

The manuscript “Calcium-Mediated Cell Wall Remodeling and MAPK/cAMP Signaling Coordinately Regulate Low-temperature Acclimation in Schizophyllum commune” addresses a relevant question for understanding the physiological responses of basidiomycete fungi to low temperature. The combination of assessments of growth, development, gene expression, and cellular indicators has the potential to contribute scientifically. However, substantial revisions are required. Discrepancies between the reported results and figures, missing protocols for important assays, and mechanistic interpretations that exceed the evidence presented were identified. The comments below concern the main manuscript.

Title and Abstract

Lines 2–4: The title states that calcium-mediated cell wall remodeling and MAPK/cAMP signaling coordinately regulate acclimation. The experiments presented assess parallel responses but do not demonstrate the functional relationships among these components. Revise the title to reflect the scope of the evidence or provide direct validation of the proposed coordination.

Lines 18–28: Revise the abstract after clarifying the discrepancies in Figures 2B, 3A, and 4B. Distinguish increases in transcripts associated with a pathway from evidence of its functional activation. The statements that calcium is “essential” and that the mechanisms act in a coordinated manner require additional evidence or more cautious wording.

Lines 29–31: Present the possible applications to mushroom cultivation as perspectives. The study did not evaluate interventions to improve productivity or cultivation performance.

Introduction

Lines 36–48: The introduction contextualizes the species and its relevance, but the discussion of methane production requires a more direct connection to the research question. Consider shortening this passage and highlighting the relevance of the strain’s marine subsurface origin to the study of cold responses.

Lines 36–39: Check the suitability of references [1,2] for the statement about the sequenced genome and established genetic resources. Reference [5], which addresses genome sequencing of the species, appears more directly relevant to this context.

Lines 49–81: The overview of cold-tolerance mechanisms is broad but should distinguish responses to low temperatures above freezing from mechanisms related to ice formation. Specify which processes underpin the hypotheses actually tested at 10 °C.

Lines 58–60: Check reference [13], which is used to support the discussion of antifreeze proteins. The bibliographic entry provides the authors and title of a study on biomass estimation in Lentinula edodes, but the DOI corresponds to another paper. Identify which publication was actually used and correct the entire entry.

Lines 69–79: Replace “(Zhang et al., 2021)” with the corresponding numerical citation [9] and verify its presence in the reference list. Also correct the spacing in “genes[21, 22]” and “[25-30].Although”.

Lines 82–90: The objectives are presented clearly. However, explicitly state the central hypothesis and the distinctive contribution relative to previous studies on fungal cold responses. Distinguish physiological characterization from the exploratory investigation of signaling pathways.

Materials and Methods

Lines 93–107: Provide additional culture conditions: MM composition and pH, preculture age and conditions, vessel volume, working volume, agitation, lighting, and timing of transfers between temperatures. Previous references are useful, but essential experimental parameters should be accessible in the manuscript or supplementary material.

Lines 98–107: Describe how relative growth and biomass were determined. Specify whether colony diameter, radius, or expansion rate was used, whether the initial inoculum diameter was subtracted, and whether biomass represents fresh or dry weight. For dry weight, provide the washing, drying, and weighing procedures.

Lines 103–105: Report the solvent, final vehicle concentration, and corresponding control for U0126, together with the rationale for the dose used. Describing it as a specific inhibitor of all MAPK signaling is overly broad without target validation in S. commune.

Lines 109–113: Describe spore collection, observation intervals, the number of spores evaluated, and the operational criterion for germination. Clarify the relationship between the length measurement mentioned in the Methods and the germination time presented in the Results.

Lines 114–117: The protocol describes initial growth at 30 °C but does not clarify when and how the 10 °C treatment was established. Report light intensity, humidity and ventilation conditions, criteria for identifying reproductive stages, and the starting point for measuring the times presented in Figure 2A.

Lines 119–122: Detail chitin quantification: sample preparation, fresh- or dry-weight basis, hydrolysis temperature and duration, colorimetric reaction reagents, standard, calibration curve, and final calculation. Justify the suitability of the adaptation of method [37] to the fungal matrix and check the unit presented in Figure 3A.

Lines 124–132: Supplement the RT-qPCR protocol with the equipment, RNA quantity and quality, reaction volume, primer concentrations, amplification conditions, negative controls, and reaction specificity and efficiency.

Lines 91–139; results in lines 201–224 and 268–272: Include the currently undescribed protocols for ROS, MDA, GSH/GSSG, GSH-Px, SOD, total antioxidant capacity, and calcium. Report extract preparation, methods or kits, manufacturers, equipment, measurement conditions, calibration, and normalization. For results expressed per amount of protein, describe protein determination. For calcium, clarify whether the assay quantifies total calcium associated with biomass or free intracellular calcium; these measurements are not equivalent.

Lines 134–139: Supplement the microscopy description with the equipment, acquisition conditions, number of fields and independent samples, image selection criterion, and staining quantification method. Include appropriate controls for interpreting Evans blue uptake.

Lines 141–149: Revise the statistical analysis. The LSD test does not, by itself, provide a solution for unequal variances. Report the assumptions assessed, the tests applied to each dataset, and how multiple comparisons were controlled. For Figure 4, consider a model that evaluates temperature, time, and their interaction, according to the sample structure.

Results

Lines 163–168: The caption mentions letters indicating statistical differences, but the figure uses asterisks. Adjust the caption to match the elements actually presented.

Lines 170–181 and Figure 2: Resolve the discrepancy between the text and Figure 2B. The text describes a delay in germination from 18 h to 74 h under low temperature, whereas the graph associates approximately 18 h with 10 °C and 74 h with 30 °C.

Lines 183–199 and Figure 3A: The text states a 59.42% increase in chitin content at 10 °C, but the graph shows a lower value at 10 °C and a higher value at 30 °C. This discrepancy compromises the interpretation of cell wall remodeling based on chitin accumulation. Check the original data, labels, and percentage calculation. After clarification, discuss chitin content and CHS gene expression together, without treating increased transcript levels as a direct measure of the synthesis rate.

Lines 201–224 and Figure 4: Figure 4B shows lower MDA at 10 °C than at 30 °C at both time points, contradicting the statement in lines 203–205. Furthermore, the panel does not show the reduction in MDA between 24 h and 96 h at 10 °C described in lines 209–210. Check the data, labels, and interpretation.

Lines 249–266 and Figure 6: Correct the repetition of PKA1 in the descriptions of the highest and intermediate expression levels, checking whether one of these mentions should refer to PKAR. The graph does not display the statistical groupings used in the comparisons mentioned in the text.

Lines 268–293 and Figure 7: The increase in measured calcium and the staining following EDTA treatment do not, by themselves, demonstrate that calcium acts as an upstream regulator of cell wall remodeling. Evans blue provides information on compromised cellular integrity/permeability but does not specifically identify cell wall damage. The staining also does not demonstrate direct chelation of intracellular calcium. To support this interpretation, specificity controls, calcium replenishment, and direct cell wall measurements under the treatments would be required. Add scale bars to the micrographs and report the number of replicates and quantification. Correct the fragment “Different lowercase letters.”, since the panel displays asterisks.

Discussion

Lines 295–319: Check references [45,46]: reference [46] addresses epigenetic regulation in S. commune and does not directly correspond to the set of species and temperature effects mentioned in lines 301–303.

Lines 320–337: Reassess the arguments concerning chitin accumulation and recovery of redox status after resolving the discrepancies in Figures 3A and 4B. The hypothesis of investment in the cell wall at the expense of growth is interesting but was not directly tested.

Lines 338–356: Check references [49,50], which are used to support MAPK functions in Aspergillus fumigatus and Magnaporthe oryzae. The listed entries concern Antarctic yeast adaptation and cold-active lipases/esterases, respectively. Also review the relevance of reference [51] to the statement about cAMP-PKA. The qualification in lines 354–356, acknowledging the need to measure cAMP and PKA activity, is appropriate and should also be reflected in the title, abstract, and results.

Lines 357–365: Distinguish the study findings from the proposed mechanistic model. The position of calcium upstream of the other responses, the interactions between pathways, and the effect of EDTA on chitin and CHS expression were not demonstrated.

Conclusion

Lines 366–380: The manuscript presents a synthesis at the end of the discussion, without a separate conclusion section. The central point is that this synthesis should address the objectives based on confirmed results, without reiterating mechanisms that have not been demonstrated.

References

Lines 423–424, reference [13]: The entry combines two distinct papers. The article by Jomura et al. on Lentinula edodes biomass has the DOI 10.1371/journal.pone.0232049. The DOI provided in the manuscript corresponds to a study on antifreeze proteins. Correct the entry according to the publication actually used.

Lines 425–427: Correct the numbering of the reference by Weinstein et al., which is presented as “1.” in the position corresponding to reference [14].

Lines 124–125 and the list of abbreviations after line 398: Adjust the expansion of RT-qPCR to indicate the reverse transcription step and correct “Citin” to “Chitin”.

Language revision and units: Standardize gene nomenclature, units, spacing between values and °C, and expressions such as “low-temperature stress”. Correct constructions such as “Low-temperatures stress”. Language revision should accompany the correction of scientific inconsistencies.

Author Response

Comments 1:

Response 1: We agree with this comment. The revised title is: "Calcium-Associated Cellular Integrity and MAPK/cAMP Signaling are Associated with Low-temperature Acclimation in Schizophyllum commune 20R-7-F01".

Comments 2:

Response 2: We agree with this comment. We have carefully reviewed and revised Figures 2B, 3A, and 4B to ensure the accuracy and consistency of data presentation, labeling, and statistical information. The revised figures have been incorporated into the modified manuscript. The abstract has also been revised with more precise wording; please refer to the modified manuscript for details. Line: 25-30.

Comments 3:

Lines 29–31: Present the possible applications to mushroom cultivation as perspectives. The study did not evaluate interventions to improve productivity or cultivation performance.

Response 3:

We thank the reviewers for their valuable comments. We fully agree that this study did not evaluate interventions aimed at increasing yield or improving cultivation performance, and thus the potential applications in mushroom cultivation should not be overstated. In accordance with the reviewers' request, the content regarding potential applications in mushroom cultivation has been removed from the manuscript.

Comments 4:

Response 4:

We agree with this comment. The description of methane in the introduction has been removed. Additionally, a description has been added regarding the importance of the strain's origin deep-sea sediments to the study of its low-temperature response. The added content is as follows: More importantly, the S. commune 20R-7-F01 used in this study was isolated from deep-sea sediments, an environment characterized by extreme conditions year-round. This unique origin makes it an ideal model for investigating the molecular mechanisms of low-temperature adaptation.

Comments 5:

Response 5:

We agree with this comment. Here, we have added reference [3] to support the statement regarding genome sequencing, while the original references [1, 2] support the statement concerning existing genetic resources. Line: 39.

Comments 6:

Response 6:

We agree with this comment. The introduction has been revised in accordance with the suggestions to clearly distinguish between "low-temperature responses above freezing" and "mechanisms associated with ice crystal formation." Furthermore, we have explicitly stated that the cAMP, MAPK, and Ca²⁺ signaling pathways, along with cell wall remodeling and antioxidant responses, form the theoretical basis for the hypotheses experimentally tested in this study at 10 °C.

The additional content is as follows:

It is important to distinguish between responses to low-temperature above freezing and mechanisms specifically related to ice formation. The former primarily involves metabolic and signaling adjustments to maintain cellular function under low-temperature conditions, whereas the latter includes antifreeze proteins and ice-recrystallization-inhibiting substances that mitigate mechanical damage caused by ice crystals. Line: 55-59.

Among these, the cAMP, MAPK, and Ca²⁺ signaling pathways, together with cellular integrity and antioxidant responses, represent the primary processes hypothesized to operate at 10 °C in this study. Line: 84-86.

Comments 7:

Response 7:

We agree with this comment. The entire reference entry has been carefully checked and corrected. Line: 656-657.

Comments 8:

Response 8:

We agree with this comment. It has been verified and corrected. Line: 76-84.

Comments 9:

Response 9:

We agree with this comment. It has been revised; the current wording is: In this study, we investigated the physiological and molecular responses of S. commune 20R-7-F01 to low-temperature stress. We hypothesize that this deep-sea derived strain possesses unique low-temperature mechanisms, and that its physiological and molecular responses to low-temperature are associated with MAPK, cAMP, and Ca²⁺ signaling pathways. To test this hypothesis, we first performed a systematic physiological characterization of low-temperature responses, including the effects of low-temperature exposure on mycelial growth, fruiting body development, and basidiospore germination, as well as low-temperature induced changes in chitin content and CHS gene expression, oxidative-stress biomarkers, and antioxidant capacity. On this basis, we further conducted an exploratory investigation of the signaling pathways potentially involved in low-temperature adaptation using transcript analysis and pharmacological treatments, with a focus on MAPK, cAMP, and Ca²⁺ associated signaling responses. This study elucidates the potential mechanisms by which the S. commune 20R-7-F01 responds to low-temperature stress. Unlike previous studies that primarily focused on the low-temperature response of mesophilic or terrestrial fungi, this research offers a unique perspective by linking the low-temperature adaptation strategies of a deep-sea derived basidiomycete to its distinctive ecological origin, thereby deepening our understanding of environmental adaptation in basidiomycetes.

Comments 10:

Response 10:

Detailed revisions have already been made in sections 2.1 and 2.2. Line: 109-140.

Comments 11:

Response 11:

We agree with this comment. The methods for determining relative growth and biomass have been added to sections 2.3 and 2.4. Line: 144-161.

Comments 12:

Response 12:

We agree with this comment. The relevant content has been revised in section 2.2. Line: 119-140.

Comments 13:

Response 13:

We agree with this comment. This content has been supplemented in section 2.5. Line: 162-197.

Comments 14:

Response 14:

We agree with this comment. This content has been supplemented in section 2.5. Line: 162-197.

Comments 15:

Response 15:

We agree with this comment. Detailed steps for the quantitative analysis of chitin have been added to section 2.6 (Line: 198-209). This method is independent of specific fungal species; the experimental standard deviation is less than 5%, demonstrating good precision and applicability. The units in Figure 3A have been verified and corrected: μg g^-1 FW represents the chitin content per g of fresh weight.

Comments 16:

Response 16:

We agree with this comment. This content has been supplemented in section 2.9. Line: 241-264.

Comments 17:

Response 17:

We agree with this comment. This content has been supplemented in section 2.7 and 2.8 (Line: 210-240). The calcium content within the mycelium was determined.

Comments 18:

Response 18:

We agree with this comment. This content has been supplemented in section 2.11. Line: 276-291.

Comments 19:

Response 19:

We agree with this comment. This content has been supplemented in section 2.13. Line: 306-319.

Comments 20:

Lines 163–168: The caption mentions letters indicating statistical differences, but the figure uses asterisks. Adjust the caption to match the elements actually presented.

Response 20:

We agree with this comment. Corrections have already been made in the appropriate places. Line: 334-338.

Comments 21:

Response 21:

We agree with this comment. We have carefully reviewed the issue, made the necessary corrections, and resubmitted the images. Line: 349.

Comments 22:

Response 22:

We agree with this comment. We have carefully reviewed the issue, made the necessary corrections, and resubmitted the images. We have added this paragraph at the end of 3.3: However, it should be noted that the upregulation of CHS transcripts does not necessarily reflect a corresponding increase in chitin synthesis rate, as chitin content is determined by the balance between synthesis and degradation. Therefore, these data should be interpreted as evidence of transcriptional activation and chitin accumulation rather than a direct measure of chitin biosynthesis rate. Line: 363-367.

Comments 23:

Response 23:

We agree with this comment. We have carefully reviewed the issue, made the necessary corrections in section 3.4, and resubmitted the images. Line: 393.

Comments 24:

Response 24:

We agree with this comment. We have carefully reviewed the issue, made the necessary corrections in section 3.6, and resubmitted the images. Line: 438-439.

Comments 25:

Response 25:

We agree with this comment. In the revised manuscript, we have added experimental results regarding direct calcium chelation and data on cell wall integrity observed via transmission electron microscopy (TEM). We have redrawn Figure 7, adding scale bars to the micrographs, providing quantitative analysis of the Evans blue staining results (Figure 7C), and indicating the number of experimental replicates. The relevant TEM methodology has been included in the "Materials and Methods" section (2.12), and the statement regarding "different lowercase letters" has been corrected.

Comments 26:

Response 26:

We agree with this comment. We have selected the references to support this section. Line: 507-509.

Comments 27:

Response 27:

We agree with this comment. This section has been revised, and the current description is: This phenomenon may represent a compensatory response that helps maintain structural stability when cellular growth is restricted. However, this study did not directly verify whether this cell wall maintenance strategy comes at the expense of growth. Therefore, the observed changes suggest a possible trade-off between cell wall maintenance and fungal growth that warrants further investigation.

Comments 28:

Response 28:

Thank you for your constructive suggestions. We have carefully re-evaluated and replaced the references with more appropriate ones (Line: 551-552). Additionally, we have revised the relevant statements in the title, abstract, results, and discussion sections to ensure consistency with our experimental evidence. In particular, we have adjusted the wording to reflect the fact that, while our data show elevated intracellular cAMP levels, PKA activity was not directly measured (Line: 562-564).

Comments 29:

Response 29:

We agree with this comment. We have revised the relevant content to clearly distinguish between the experimental results and our proposed mechanistic explanations. Specifically, we have removed or qualified statements suggesting that Ca²⁺ acts upstream of the MAPK/cAMP signaling pathways or directly regulates chitin and CHS expression. We now explicitly state that the potential interactions among Ca²⁺, MAPK, and cAMP signaling pathways as well as the effects of Ca²⁺ depletion on chitin accumulation and CHS expression have not been directly confirmed in this study and require further experimental validation.

The current wording is as follows: Ca²⁺ signaling provides another potential mechanism linking low-temperature perception with cellular protection. Mycelial Ca²⁺ content increased by 30.61% under low-temperature conditions, whereas Ca²⁺ depletion resulted in impairment of cellular integrity (Figure 7). Ca²⁺ is an important second messenger involved in fungal stress responses and cell wall regulation [60]. The increased Ca²⁺ content observed here is consistent with a possible role of Ca²⁺ signaling in maintaining cellular integrity under low-temperature stress. However, whether Ca²⁺ acts upstream of MAPK or cAMP signaling, and whether these pathways interact under low-temperature stress, remains to be determined. Likewise, the effects of Ca²⁺ depletion on chitin content and CHS expression were not directly examined in this study. Together with the MAPK and cAMP responses, Ca²⁺ signaling may be part of a broader regulatory network associated with cellular adaptation to low-temperature, although the relationships among these pathways require further experimental validation.

Comments 30:

Response 30:

We agree with this comment. This section has been revised in accordance with the reviewers' suggestions.

Comments 31:

Response 31:

We agree with this comment. The DOI for this reference has been corrected based on the literature actually cited. Line: 656-657.

Comments 32:

Lines 425–427: Correct the numbering of the reference by Weinstein et al., which is presented as “1.” in the position corresponding to reference [14].

Response 32:

We agree with this comment. The reference number has been corrected. Line: 658-660.

Comments 33:

Lines 124–125 and the list of abbreviations after line 398: Adjust the expansion of RT-qPCR to indicate the reverse transcription step and correct “Citin” to “Chitin”.

Response 33:

We agree with this comment. All adjustments have been made. Line: 631.

Comments 34:

Response 34:

We agree with this comment. The entire text has been verified, and all necessary revisions have been made.

Reviewer 4 Report

The manuscript by Leng et al. reports the effect of low temperature on Schizophyllum commune basidiospore germination, vegetative growth and fruitbody formation. Low temperature (10⁰) inhibited all these processes. Indeed, S. commune likes to grow at high temperature (30⁰-35⁰) and light and ventilation leads to fruitbody formation at 25⁰-30⁰. On other hand the mycelium can be kept alive at 4⁰ for years if the moisture is kept. The novel results in the manuscript are that the cAMP and calcium plays a role in the development of low temperature tolerance in S. commune. The effect of low temperate on the development in S. commune might not be a universal property of basidiomycetes (Agaricomycetes) as the authors at the end of the introduction suggest “ This study provides new insights into the mechanisms underlying cold stress responses in S. commune and contributes to a better understanding of environmental adaptation in basidiomycete fungi.“ In northern hemisphere the fruitbodies of many basidiomycetes develop at fall when the temperature drops, although the shortening of day and moisture also affects. During cultivation of Agaricus bisporus drop of temperature to 15⁰ is used to induce fruitbody development. The manuscript should emphasize that there are plenty of variation among fungi in respect to low temperature.

The following sites need attention:

line 98 The growth of S. commune 20R-7-F01 was/ the growth of dikaryotic S. commune

line 109 Basidiospores were collected from fruitbodies of S. commune 20R-7-F01

133 Evans blue staining for what purpose

Fig.3A the columns do not fit the text

Results

Chitin synthetase genes, ROS-pathway genes, genes of MAPK- and cAMP signaling pathways were selected (chosen) on what basis? On the basis of the general knowledge of genes known to be involved in fungal development/ or at low temperature (?). The expression of chosen genes was estimated by RT-qPCR from isolated RNA-samples of each growth condition. Could it be that some important genes escaped attention?

Discussion

lines 301-303 “These effects are consistent with previous observations in Pleurotus ostreatus, Lentinula edodes, and Flammulina velutipes, in which suboptimal temperatures reduced mycelial growth and delayed reproductive development [45, 46]".

There is some discrepancy between the text and references 45, 46. No Flammulina velutipes is mentioned. in Pleurotus ostreattus the best biological efficiency (most fruitbodies produced ) was obtained from cultures grown at 15^0..

See my statement to authors

Author Response

Comments 1:

The manuscript by Leng et al. reports the effect of low temperature on Schizophyllum commune basidiospore germination, vegetative growth and fruitbody formation. Low temperature (10⁰) inhibited all these processes. Indeed, S. commune likes to grow at high temperature (30⁰-35⁰) and light and ventilation leads to fruitbody formation at 25⁰-30⁰. On other hand the mycelium can be kept alive at 4⁰ for years if the moisture is kept. The novel results in the manuscript are that the cAMP and calcium plays a role in the development of low temperature tolerance in S. commune. The effect of low temperate on the development in S. commune might not be a universal property of basidiomycetes (Agaricomycetes) as the authors at the end of the introduction suggest “This study provides new insights into the mechanisms underlying cold stress responses in S. commune and contributes to a better understanding of environmental adaptation in basidiomycete fungi. “In northern hemisphere the fruitbodies of many basidiomycetes develop at fall when the temperature drops, although the shortening of day and moisture also affects. During cultivation of Agaricus bisporus drop of temperature to 15⁰ is used to induce fruitbody development. The manuscript should emphasize that there are plenty of variation among fungi in respect to low temperature.

Response 1:

We agree with this comment. We expanded the Discussion to emphasize that low-temperature responses vary among fungal species, developmental stages, and environmental conditions. We now state explicitly that the results obtained with the deep-sea-derived S. commune 20R-7-F01 strain should not be generalized to other basidiomycetes or fungi. Line: 593-601.

Comments 2:

line 98 The growth of S. commune 20R-7-F01 was/ the growth of dikaryotic S. commune

Response 2:

We agree with this comment. It has been clarified that the experimental strain is a dikaryotic strain. Line: 114.

Comments 3:

line 109 Basidiospores were collected from fruitbodies of S. commune 20R-7-F01

Response 3:

We agree with this comment. This description has been supplemented. Line: 163-164.

Comments 4:

133 Evans blue staining for what purpose

Response 4:

We agree with this comment. Additional explanation regarding the purpose of Evans blue staining has been provided. Line: 280-282.

Comments 5:

Fig.3A The columns do not fit the text

Response 5:

We agree with this comment. The descriptions in the text have been verified, and Figure 3 has been resubmitted. Line: 368.

Comments 6:

Response 6:

Thank you for this important comment. The genes analyzed in this study were selected based on previously established knowledge of fungal development, cell wall integrity, oxidative stress responses, and signal transduction pathways, with particular consideration of their reported involvement in fungal growth and responses to environmental stress, including temperature stress. Specifically, chitin synthase genes were selected because chitin biosynthesis is essential for fungal cell wall formation and hyphal growth; ROS-related genes were selected because oxidative stress and ROS homeostasis are closely associated with fungal responses to environmental changes; and representative genes involved in the MAPK and cAMP signaling pathways were selected because these pathways are well-established regulators of fungal development, stress responses, and cellular adaptation.

Thus, the RT-qPCR analysis was designed as a candidate-gene-based approach to examine whether these major biological processes and signaling pathways are associated with the observed growth and physiological responses under the tested conditions. Total RNA was isolated from mycelia grown under each experimental condition, and the expression levels of the selected genes were determined by RT-qPCR.

We agree with the reviewer that, given the complexity of fungal regulatory networks, it is possible that other genes involved in fungal development, low-temperature responses, or stress adaptation were not included in our analysis. The selected genes therefore do not represent an exhaustive survey of all genes potentially involved in these processes. A transcriptome-wide analysis would be required to comprehensively identify additional genes and pathways that may respond to the tested conditions.

Comments 7:

Response 7:

We agree with this comment. We have selected the references to support this section. Line: 507-509.

Round 2

Reviewer 2 Report

The authors have addressed all of the issues.

None

Reviewer 3 Report

Dear Authors,
Thank you for carefully revising the manuscript and addressing my comments. The revisions have improved the clarity of the title, methods, the consistency of the results, and the alignment of the discussion with the evidence presented. I am satisfied with the changes and have no further comments. I recommend acceptance of the revised manuscript.

Reviewer 4 Report

In the manuscript there is lot changes that are done probably on the basis of the statement by Reviewer 2, whose statement I have not seen. The changes done based on the statement of Reviewer 2 improve and add details to manuscript. Of course they have to be accepted by Reviewer 2. I stand only behind my comments.

See my comment above.