Deciphering the ceRNA Network in Alfalfa: Insights into Cold Stress Tolerance Mechanisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Biomolecules Manuscript #:  3998390

Authors:  Lin Zhu et al., 2025

Title:  Deciphering the ceRNA Network in Alfalfa: Insights into Cold Stress Tolerance Mechanisms

 

The authors have use genomic, transcriptomic and bioinformatic analysis of alfalfa (Medicago sativa) to predict RNAs and genes that associate with cold stress tolerance.  They identify quite a few RNAs (mRNAs, microRNAs, long non-coding RNAs, and circular RNAs) with differential expression in response to cold stress treatment.  From this, and the predict gene function (via GO and KEGG), they make predictions of metabolic, cellular, and molecular processes altered in cold-stressed plants. They also used a SNP-based genome-wide associate study to map loci that statistically associate with cold tolerance, and from this they predict that the gene MS.gene53818 that encodes the alfalfa UDP-arabinopyranose mutase 1 (UAM1) thought to be involved with arabinose containing polysaccharides in the cell wall.  Further, with their proposed competing endogenous RNA (ceRNA) network, they predict two genes (Ms.gene049457 and MS.gene41592) thought to encode WRKY33 plant transcript factors, among other predictions.

Understanding plant acclimation and adaptation to cold stress is important in alfalfa that has to overwinter, along with other plant species.  And, their approach is a logical one using solid genomic, transcriptomic, and bioinformatics approaches, and it yields what seems to be reasonable and convincing predictions.  What is lacking are data that goes the next step to show direct evidence that indeed the predictions identify functional genes/proteins that do increase cold tolerance.  As is, the predictions are still speculative, as convincing as the bioinformatics predictions are.  They provide some argument in the Conclusion that the WRK33 predicted transcription factors do have function in cold tolerance by citing work from other sources/literature in other plant species.  That is fine, but direct evidence for functionality is needed to give this manuscript a higher impact and be more convincing that would show in fact one or two of the key identified candidate genes directly provide cold tolerance in alfalfa.  These data could come from different types of experiments.  A direct knock-out mutant for each of the key candidate genes that then shows reduction in cold tolerance would be ideal.  However, these might be very time-consuming and difficult to obtain.  Alternative, perhaps mutants are known and characterized for these genes in other plant species, that the authors could reference to build the argument that these genes have direct function in cold tolerance.  Note, there is a UAM gene mutant (mur5) in Arabidopsis that has reduced arabinose in cell well that could be tested for cold tolerance acclimation, which Arabidopsis has (C. Dugard et al., 2016 Plant Physiology. Vol 171, pg 19056-1920).  Or, at least this should be mentioned and discussed.  Another option for direct support would be more evidence of genetic variations (alleles/SNPs) or significant variation in gene expression for candidate genes/RNAs among multiple cold-tolerant cultivars versus multiple non-cold-tolerant cultivars.  The authors mentioned this strategy in their manuscript (Introduction Lines 79 -80), but according to the Methods they only tested one cultivar “Zhaodong” with cold-treatment (LD) and non-cold-treatment (YM).  Some of the Methods was missing critical information (see below) about cultivars, so it is possible they already have these data but just not clear in the written manuscript.

The reason the above issue about direct evidence of gene/protein functioning in cold tolerance is so important is because for all the predicted changes in RNA expression (mRNA, micrRNA, etc…) they could be direct (primary) cold acclimation responses or they could be changes that are indirect (secondary) response to damage due to cold. The latter is useful, but different than genes/RNAs/proteins with roles in cold acclimation. 

In addition to the above issue (predictions being speculative and needing direct functional evidence for cold tolerance), there are some key writing issues and missing information in the Methods that makes it genuinely hard to understand exactly what was done, what cultivars were used and/or treated. These writing issues makes it harder to interpret the conclusions, and this is the main reason that I marked the "Quality of Presentation" as being low.  In general, what is needed is more information about exactly what cultivars of alfalfa were used for each of the genomic and transcriptomic experiments.  Under the section “Plant Materials and Treatments” within Methods, only one cultivar (Zhaodong) is mentioned regarding treating with cold or non-cold treatments. And, even for that, it was not clear what the LD and YM treatments were (see below). In same section of Methods, the second paragraph indicated that “Leaves from 291 alfalfa accessions were collected”, but there was no additional information provided about these accessions in either the manuscript or the supplemental documents (23 Supplemental Tables and 3 Supplemental Figures).  The Introduction mentions (Lines 97 – 80) they tested “two alfalfa cultivars with completely opposite cold resistance…”, but what the second cultivar is was never mentioned nor were there any clear data/experiments in manuscript or supplemental data that describes or characterizes these.

Section 2.3 (DNA extraction, sequencing, etc..) has the same issues. The authors mention that DNA was extracted from the leaves using a specific method, which is needed.  But, again, no information about what specific plants, cultivars, or accession (neither names nor ID numbers/links), etc… were used.  Where these the 291 plants mentioned above?  Where these the “300 alfalfa accessions” mentioned in Results (Line 282), Section 3.2 (GWAS identifies significant SNPs …)?  Again, no details or information was provided about these.  Why would this number not be 291, from Methods section?  No details provided, which are required.

            In addition to this concern, there a number of writing issues/typos/formatting issues that I feel the authors need to resolve before this could be published.  See my specific comments below for these issues.

 

Abstract:

Lines 38:  delete extra space in “alfalfa’s”

 

Introduction:

Line 45:  Delete “s”, so that it reads, “…OsG1 can affect the expression…”

Lines 58 - 59:  Sentence does not make sense, seems circular logic.  It says, in essence, “sucrose is first transformed into starch and raffinose.  Then, starch is transformed into sucrose.”  Again, circular logic, unless the meaning is something else.  Please rewrite to be clear.

Line 59: near end, it says, “Therefore, function genes …”. Should be either “functional” or something different.  OR, better yet, delete “function”, for it’s not needed for what is “non-functional gene?”

Line 79.  This was mentioned above in general concerns/issues.  Authors state that "two cultivars with completely opposite cold resistance” were tested. Yet, only one cultivar (Zhaodong) is ever mentioned specifically throughout the entire manuscript. See above for this issue and other problems related to missing information in Methods.

 

Methods:

Line 85:  Needs a reference or supporting data to show that the cultivar Zhaodong has “excellent cold resistance”.

Lines 85 – 91:  This paragraph is unclear and confusing.  First, define what LD and YM stand for, aside from cold treated and control.  Next, the last sentence makes it seem that both LD and YM were treated with a 16/8 light photoperiod AND 4oC for 24 hours.  I can only guess that only the LD was treated with the 4oC for 24 hours. But, what was the standard temperature for the control and outside of the 4oC 24 hour period for LD?  These details are critical to understand experiment used to obtain RNAs for differential expression studies that are absolutely central to this manuscript.

Line 92:  Also as mentioned above.  It says, “Leaves of 291 accessions were collected and how they were grown and that cold resistance was evaluated. But at no point are the accessions every mentioned nor do the authors ever show data about the “cold resistance” assessments and how used for these experiments.  Details about the accessions, their names/ID’s and links to sources for them, as well as data on the cold resistance assessment, etc....

Line 110:  species name Medicago sativa needs to be italicized, along with all the other plant species names included in the manuscript Results and Discussion/Conclusion sections.

Line 114.  Says DNA was extracted from leaves, but from what cultivars, how/where grown?  Are these the 291 accessions mentioned in Line 92 or something else?  Or, were these the 300 accessions mentioned on Line 282 in Results?  Must be explained.

Line 128:  They used edgeR to determine DE RNAs “…between the treatment and control groups”.  Are these the LD and YM?  If so, then please state that.  If not, explain otherwise.

Line 137:  Same as for Line 128, explain what samples and/or cultivars were used for the sRNA sequencing were used for Methods section 2.5.  Incomplete information.  And same goes for Methods Sections 2.6, 2.7, and 2.8.

Line 167:  Typo in heading, should be, “…circRNA” (not cricRNA)

 

Results:

Line 192:  Similar to issues above with Methods, not enough information is provided to understand what is being tested/studied.  Here the authors state that “six cDNA libraries”, including those from control YM and cold-treated LD.  My guess (from looking through data and supplemental tables) is they mean are six cDNA libraries from Zhaodong cultivar, three are replicates from LD cold-treated plants and the other three are replicates from YM control treated plants.

Line 200:  The authors “state six databases”, and my guess is they meant data from the six cDNA libraries.  As is, it implies they downloaded data from six different genomic/transcriptomic databases that are not mentioned or referenced anywhere.

Figure 1B:  The GO categories are impossible to read due to tiny font size. These must be increased in size for anyone to read.  If not, reformat to make these clear and readable in some other way.

Lines 234 – 237:  This statement relates to the comment made above in general issues about whether changes in RNA expression are direct (primary) cold acclimation responses or are the changes an indirect (secondary) response to damage due to cold?

Line 250:  To say these responses “work together” is speculative, at this point.  Sharing intermediates and materials does not necessarily mean they “work together”.

Lines 258-274:  The very broad list of GO and KEGG identified biochemical pathways is so wide ranging (from general Transcription Factors to Alkaloid biosynthesis pathways to plant hormone signals that it is truly hard to take any specifics away related to cold tolerance. That list could describe many different stress and non-stress responses in plants. My point being, this is very speculative and one should be cautious about making too specific a prediction from these, at this point.

Line 282:  As mentioned above several times, no specific information is provided anywhere about these 300 alfalfa accessions.  Are these the 291 mentioned in Line 92?  Different ones? These must be explained.

Lines 299-300: This first sentence is of such general knowledge; the sentence can be deleted.

Lines 340 – 341:  Do the authors suggest the alkaloids are functioning as primary metabolites to help the plants or would these alkaloids be secondary “defense” metabolites?  Plants can produce thousands of different types of alkaloids.

Line 356:  Should be singular, “transcription”

Line 394: Typo in heading, seems it should be, “DEcircRNA” (not DElncRNA).

Line 419:  Again, what cultivars and/or treated samples used here for ceRNA network predictions?

Line 441:  Since the ceRNA network is a prediction, which seems supported, conclusions from it would be predictions.  So, to say the ceRNA network can enhance seems a stretch.  Instead, it would seem better to say these “may enhance” or “are likely involved”.

 

Discussion:

Line 472:  conversion of starch and sucrose to what?  Or, are they saying conversion of “starch to sucrose”?  Rewrite to clarify meaning.

Lines 478-479:  The authors could strengthen their argument that MsUAM1 is involved in cold acclimation by pointing out that it showed a significant increase in expression under cold treatment, as they mentioned previously (Lines 291-292 and Table S3).

            This is also a section that they should add any information about susceptibility to cold stress in Arabidopsis mutant mur5 (as mentioned above; C. Dugard et al., 2016 Plant Physiology. Vol 171, pg 19056-1920).

Lines 503 – 507:  Several plant species names need to be italicized.

Line 559:  As mentioned above, the predicted ceRNA network is a prediction. So, conclusions from this are also predictions.  Thus, should say, “how crRNAs might regulate alfalfa cold tolerance…”.

 

Conclusions:

Line 572:  UAM1 and WRKY33 gene names need to be italicized.

 

In general, as mentioned above, these are all reasonable predictions.  But, what is lacking are data that goes the next step to show direct evidence that indeed the predictions identify functional genes/proteins that do increase cold tolerance.  Alfalfa mutants with individual gene knock-outs or to test published / known mutants in other plants (such as the mentioned mur5 Arabidopsis), etc…. Direct evidence of cold-tolerance acclimation for the key genes would help this manuscript be more impactful.

 

Supplemental Tables and Figures:

Table S8 has not useful information.  All data state, “#N/A”

Comments on the Quality of English Language

Nothing in addition to what was mentioned above.

Author Response

We would like to extend our sincere gratitude to the anonymous SCI reviewers for their generous investment of time, expertise, and thoughtful effort in evaluating this manuscript. Their meticulous feedback, ranging from nuanced suggestions on experimental methodology validation to targeted guidance on clarifying molecular mechanism interpretations and streamlining data presentation, has been instrumental in elevating the rigor and clarity of this work. Beyond refining the paper’s technical and narrative quality, their constructive insights have deepened my understanding of the field and sharpened our research skills. We have made serious modifications to address the following issues.

 

Comments 1:Understanding plant acclimation and adaptation to cold stress is important in alfalfa that has to overwinter, along with other plant species.  And, their approach is a logical one using solid genomic, transcriptomic, and bioinformatics approaches, and it yields what seems to be reasonable and convincing predictions.  What is lacking are data that goes the next step to show direct evidence that indeed the predictions identify functional genes/proteins that do increase cold tolerance.  As is, the predictions are still speculative, as convincing as the bioinformatics predictions are.  They provide some argument in the Conclusion that the WRK33 predicted transcription factors do have function in cold tolerance by citing work from other sources/literature in other plant species.  That is fine, but direct evidence for functionality is needed to give this manuscript a higher impact and be more convincing that would show in fact one or two of the key identified candidate genes directly provide cold tolerance in alfalfa.  These data could come from different types of experiments.  A direct knock-out mutant for each of the key candidate genes that then shows reduction in cold tolerance would be ideal.  However, these might be very time-consuming and difficult to obtain.  Alternative, perhaps mutants are known and characterized for these genes in other plant species, that the authors could reference to build the argument that these genes have direct function in cold tolerance.  Note, there is a UAM gene mutant (mur5) in Arabidopsis that has reduced arabinose in cell well that could be tested for cold tolerance acclimation, which Arabidopsis has (C. Dugard et al., 2016 Plant Physiology. Vol 171, pg 19056-1920).  Or, at least this should be mentioned and discussed.  Another option for direct support would be more evidence of genetic variations (alleles/SNPs) or significant variation in gene expression for candidate genes/RNAs among multiple cold-tolerant cultivars versus multiple non-cold-tolerant cultivars.  The authors mentioned this strategy in their manuscript (Introduction Lines 79 -80), but according to the Methods they only tested one cultivar “Zhaodong” with cold-treatment (LD) and non-cold-treatment (YM).  Some of the Methods was missing critical information (see below) about cultivars, so it is possible they already have these data but just not clear in the written manuscript.

Response 1: We have carefully addressed all of the comments raised by the editor and the reviewers, and made corresponding modifications and supplements to the manuscript.

 

Comments 2: Lines 38:  delete extra space in “alfalfa’s”

Response 2: The corresponding modifications have been made.

 

Comments 3:Line 45:  Delete “s”, so that it reads, “…OsG1 can affect the expression…”

Response 3:The corresponding modifications have been made.

 

Comments 4:Lines 58 - 59:  Sentence does not make sense, seems circular logic.  It says, in essence, “sucrose is first transformed into starch and raffinose.  Then, starch is transformed into sucrose.”  Again, circular logic, unless the meaning is something else.  Please rewrite to be clear.

Response 4: We have provided a more detailed explanation in this part.

 

Comments 5: Line 59: near end, it says, “Therefore, function genes …”. Should be either “functional” or something different.  OR, better yet, delete “function”, for it’s not needed for what is “non-functional gene?”

Response 5: We have made modifications based on this comment.

 

Comments 6: Line 79.  This was mentioned above in general concerns/issues.  Authors state that "two cultivars with completely opposite cold resistance” were tested. Yet, only one cultivar (Zhaodong) is ever mentioned specifically throughout the entire manuscript. See above for this issue and other problems related to missing information in Methods.

Response 6: First of all I would like to appologize for mixing up two cultivars with two treatment; I'm very sorry for this mistake. To avoid confusion, we have made the following modifications: In this investigation, we discovered mRNAs, miRNAs, lncRNAs, and circRNAs that respond to cold stress in the “Zhaodong” alfalfa variety and built a cold-responsive ceRNA regulatory network.

 

Comments 7: Line 85:  Needs a reference or supporting data to show that the cultivar Zhaodong has “excellent cold resistance”.

Response 7: We added these explanations in section 2.1. Plant Materials and Treatments.

 

Comments 8: Lines 85 – 91:  This paragraph is unclear and confusing.  First, define what LD and YM stand for, aside from cold treated and control.  Next, the last sentence makes it seem that both LD and YM were treated with a 16/8 light photoperiod AND  4℃ for 24 hours.  I can only guess that only the LD was treated with the 4℃ for 24 hours. But, what was the standard temperature for the control and outside of the  4℃ 24 hour period for LD?  These details are critical to understand experiment used to obtain RNAs for differential expression studies that are absolutely central to this manuscript.

Response 8: Thank you we have now included a more detailed explanation in the methods section.

 

Comments 9: Line 92:  Also as mentioned above.  It says, “Leaves of 291 accessions were collected and how they were grown and that cold resistance was evaluated. But at no point are the accessions every mentioned nor do the authors ever show data about the “cold resistance” assessments and how used for these experiments.  Details about the accessions, their names/ID’s and links to sources for them, as well as data on the cold resistance assessment, etc....

Response 9: We state this information in supplementary materials (Table S1).

 

Comments 10: Line 110:  species name Medicago sativa needs to be italicized, along with all the other plant species names included in the manuscript Results and Discussion/Conclusion sections.

Response 10: We have made the modifications accordingly.

 

Comments 11: Line 114.  Says DNA was extracted from leaves, but from what cultivars, how/where grown?  Are these the 291 accessions mentioned in Line 92 or something else?  Or, were these the 300 accessions mentioned on Line 282 in Results?  Must be explained.

Response 11: We have corrected the number of germplasm, and provided more detailed explanations.

 

Comments 12: Line 128:  They used edgeR to determine DE RNAs “…between the treatment and control groups”.  Are these the LD and YM?  If so, then please state that.  If not, explain otherwise.

Response 12: Yes, it were LD and YM, we have now provided more explicit and detailed explanations of these points in the corresponding Methods sections.

 

 

Comments 13: Line 137:  Same as for Line 128, explain what samples and/or cultivars were used for the sRNA sequencing were used for Methods section 2.5.  Incomplete information.  And same goes for Methods Sections 2.6, 2.7, and 2.8.

Response 13: Here we provide more detailed explanations.

 

Comments 14: Line 167:  Typo in heading, should be, “…circRNA” (not cricRNA)

Response 14: The text here is inaccurate. Thank the reviewer for this suggestion, this is now amended.

 

Comments 15: Line 192:  Similar to issues above with Methods, not enough information is provided to understand what is being tested/studied.  Here the authors state that “six cDNA libraries”, including those from control YM and cold-treated LD.  My guess (from looking through data and supplemental tables) is they mean are six cDNA libraries from Zhaodong cultivar, three are replicates from LD cold-treated plants and the other three are replicates from YM control treated plants.

Response 15: Yes, it was. To make this more explicit throughout the manuscript we have now described this in more detail in the methods section.

 

Comments 16: Line 200:  The authors “state six databases”, and my guess is they meant data from the six cDNA libraries.  As is, it implies they downloaded data from six different genomic/transcriptomic databases that are not mentioned or referenced anywhere.

Response 16: These library data were obtained using sequencing tools and were not downloaded from databases such as NCBI. The raw data can be obtained through communication with the corresponding authors. We have provided an explanation in the “Data Availability Statement”.

 

Comments 17: Figure 1B:  The GO categories are impossible to read due to tiny font size. These must be increased in size for anyone to read.  If not, reformat to make these clear and readable in some other way.

Response 17: Thank you, we enlarged the font in Figure 1B.

 

Comments 18: Lines 234 – 237:  This statement relates to the comment made above in general issues about whether changes in RNA expression are direct (primary) cold acclimation responses or are the changes an indirect (secondary) response to damage due to cold?

Response 18: By studying and synthesizing previous publications in the literature, we believe that changes in RNA expression are direct (primary) cold acclimation responses.

 

Comments 19: Line 250:  To say these responses “work together” is speculative, at this point.  Sharing intermediates and materials does not necessarily mean they “work together”.

Response 19: We have amended this in the text.

 

Comments 20: Lines 258-274:  The very broad list of GO and KEGG identified biochemical pathways is so wide ranging (from general Transcription Factors to Alkaloid biosynthesis pathways to plant hormone signals that it is truly hard to take any specifics away related to cold tolerance. That list could describe many different stress and non-stress responses in plants. My point being, this is very speculative and one should be cautious about making too specific a prediction from these, at this point.

Response 20: We corrected it accordingly to make this more rigorous.

 

Comments 21: Line 282:  As mentioned above several times, no specific information is provided anywhere about these 300 alfalfa accessions.  Are these the 291 mentioned in Line 92?  Different ones? These must be explained.

Response 21: We thank the reviewer for having spotted this typo of accessions number, which we corrected. We have added this additional information as supplementary material (Table S1).

 

Comments 22: Lines 299-300: This first sentence is of such general knowledge; the sentence can be deleted.

Response 22: We appreciate the reviewer’s suggestion, based on the suggestion made by the reviewers, this part was removed.

 

Comments 23: Lines 340 – 341:  Do the authors suggest the alkaloids are functioning as primary metabolites to help the plants or would these alkaloids be secondary “defense” metabolites?  Plants can produce thousands of different types of alkaloids.

Response 23: By studying and synthesizing previous publications studies, we consider that alkaloids are secondary “defense” metabolites. We apologize for being unclear on this point, that is now specified.

 

Comments 24: Line 356:  Should be singular, “transcription”

Response 24: We thank the reviewer for this careful observation and have corrected this.

 

Comments 25: Line 394: Typo in heading, seems it should be, “DEcircRNA” (not DElncRNA).

Response 25: We thank the reviewer for this careful observation and have corrected this.

 

Comments 26: Line 419: Again, what cultivars and/or treated samples used here for ceRNA network predictions?

Response 26: We thank the reviewer for this careful observation and added supplemental notes.

 

Comments 27: Line 441:  Since the ceRNA network is a prediction, which seems supported, conclusions from it would be predictions.  So, to say the ceRNA network can enhance seems a stretch.  Instead, it would seem better to say these “may enhance” or “are likely involved”.

Response 27: Followed this reviewer’s suggestion and we have correct it accordingly.

 

Comments 28: Line 472:  conversion of starch and sucrose to what?  Or, are they saying conversion of “starch to sucrose”?  Rewrite to clarify meaning.

Response 28: We are grateful for this suggestion, and now included a more detailed discussion.

 

Comments 29: Lines 478-479:  The authors could strengthen their argument that MsUAM1 is involved in cold acclimation by pointing out that it showed a significant increase in expression under cold treatment, as they mentioned previously (Lines 291-292 and Table S3).

Response 29: We have amended this in the text.

 

Comments 30:  This is also a section that they should add any information about susceptibility to cold stress in Arabidopsis mutant mur5 (as mentioned above; C. Dugard et al., 2016 Plant Physiology. Vol 171, pg 19056-1920).

Response 30: We have included a discussion of this in the revised version.

 

Comments 31: Lines 503 – 507:  Several plant species names need to be italicized.

Response 31: We have amended this in the text.

 

Comments 32: Line 559:  As mentioned above, the predicted ceRNA network is a prediction. So, conclusions from this are also predictions.  Thus, should say, “how crRNAs might regulate alfalfa cold tolerance…”.

Response 32: We have amended this in the text.

 

Comments 33: Line 572:  UAM1 and WRKY33 gene names need to be italicized.

Response 33: We have amended this in the text.

 

 

Comments 34: Table S8 has not useful information.  All data state, “#N/A”

Response 34: It should be noted that the candidate genes obtained by “#N/A” representing GWAS were not expressed in the cold stress transcriptome, and only one gene (MS.gene53818 (MsUAM1) ) was expressed and not upregulated in the cold stress transcriptome. We would like to explain why we believe this gene is a candidate gene for alfalfa cold tolerance. I adjusted the associated genes with expression levels to first place (now in Table S9).

 

 

Again, we thanks for the reviewer’s suggestions and comments!

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Recently, research has emerged on the important role of non-coding RNAs (ncRNAs) in plant responses to abiotic stress. This article explores the mechanisms by which ncRNAs and competing endogenous RNAs (ceRNAs) influence alfalfa cold tolerance. To this end, the authors conducted whole-transcriptome RNA-seq analysis and genome-wide association studies to differentially identify cold-related metabolic pathways and candidate genes, including expressed-encoded (DE) mRNAs, microRNAs, long non-coding RNAs, and circular RNAs. This study identified mRNAs and ncRNAs that may be involved in the alfalfa cold stress response, and constructed a ceRNA regulatory network associated with cold stress.
The article is well written and contains new experimental data that contribute to the advancement of the molecular physiology of stress in plants. The authors convincingly demonstrated the role of individual RNAs in the response of alfalfa plants to cold stress and identified candidate genes expressed by these RNAs. They were also able to identify metabolic pathways associated with cold stress. This study provides a foundation for further research into the molecular mechanisms underlying cold stress in plants.
There are several comments regarding the article that do not require significant revisions to the manuscript.

1. Section 2.1. Plant Materials and Treatments is not clearly written. The authors need to revise the description of the experimental setup to clarify the sequence of experiments and their purpose.

2. The experiment used an acclimation (non-damaging) temperature of 4°C, but the term "cold stress" is used throughout the article. The authors should explain in detail why they used this temperature and what effect this above-zero temperature has on cold-tolerant alfalfa.
3. Some data presented in the Supplementary Materials section may be included in the main text of the article.
4. The authors should carefully check the reference list. For example, duplicate references Nos. 23 and 70 were identified.

I believe that after these issues are addressed, the article may be accepted for publication.

Author Response

Authors of biomolecules-3998390 would like to extend our sincere gratitude to the anonymous SCI reviewers for their generous investment of time, expertise, and thoughtful effort in evaluating this manuscript. Their meticulous feedback, ranging from nuanced suggestions on experimental methodology validation to targeted guidance on clarifying molecular mechanism interpretations and streamlining data presentation, has been instrumental in elevating the rigor and clarity of this work. Beyond refining the paper’s technical and narrative quality, their constructive insights have deepened my understanding of the field and sharpened our research skills. We have made serious modifications to address the following issues.

 

Comments1: Section 2.1. Plant Materials and Treatments is not clearly written. The authors need to revise the description of the experimental setup to clarify the sequence of experiments and their purpose.

Response1: We have provided additional modifications and explanations in the revised version.

 

Comments2: The experiment used an acclimation (non-damaging) temperature of 4°C, but the term "cold stress" is used throughout the article. The authors should explain in detail why they used this temperature and what effect this above-zero temperature has on cold-tolerant alfalfa.

Response2: First of all I would like to appologize for losing symbol “-”; I'm very sorry for this mistake. We have made the necessary corrections “-4°C”.

 

 

Comments3: Some data presented in the Supplementary Materials section may be included in the main text of the article.

Response3: We have made modifications based on this comment.

 

 

Comments4: The authors should carefully check the reference list. For example, duplicate references Nos. 23 and 70 were identified.

Response4: We have made modifications based on this comment.

 

Thanks again for reviewer’s kind suggestions!

Author Response File: Author Response.pdf