Review Reports - Transcriptomic Analysis of Melatonin-Mediated Salt Stress Response in Germinating Alfalfa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Liu et al analyzed some physiological and biochemical characteristics and transcriptomes of germinating alfalfa seedlings treated with salt stress, salt stress and melatonin, together with controls. They aimed to identify the key genes and pathways of melatonin in alleviating salt stress.

The manuscript needs a thorough English check, specially the scientific editing, following examples of papers published in Agriculture, to improve the data presentation and explanation, the experiment design presentation, results and data integration, and discussion.

The figure resolution should be improved so that the figure is readable. The labels for Y-axis should be revised (there should be a space before ()). For example, Figure 1B, Y-axis, Germination rate (%); X-axis, Number of days (d); Figure 1C, Y-axis, Bud length (cm); Figure 1D, Y-axis, Fresh weight (g).

The results should be presented in Results, not expanded to Discussion (Figures 5 and 6 before 4.2).

Examples are given, but not limited to:

1. The title should be improved. One suggestion: “Transcriptomic Analysis of Melatonin-Mediated Salt Stress Response in Germinating Alfalfa”.

2. Abstract should be polished.

One suggestion: Salt stress poses a significant threat to crop yields worldwide. Melatonin (MT), an endogenous hormone synthesized in plants, has emerged as a crucial player in plant responses to various abiotic stresses, including drought, salinity, heat, and cold. However, the precise molecular mechanisms underlying MT-mediated abiotic stress responses remain incompletely understood. To elucidate the key genes and pathways involved in MT-mediated alleviation of salt stress, we conducted physiological, biochemical, and transcriptomic analyses in alfalfa seedlings. Our results demonstrated that alfalfa seedlings treated with melatonin exhibited higher germination rates, longer bud lengths, and greater fresh weights compared to those subjected to salt stress alone. Furthermore, the levels of malondialdehyde (MDA) and superoxide anion (O2-) were reduced, while the activities and contents of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and glutathione (GSH) increased in response to melatonin treatment. Transcriptome analysis revealed 2,181 differentially expressed genes (DEGs) in the salt-treated group, with 780 upregulated and 1,401 downregulated genes. In contrast, the MT-treated group exhibited 4,422 DEGs, including 1,438 upregulated and 2,984 downregulated genes. Functional annotation and pathway enrichment analysis indicated that DEGs were primarily involved in the biosynthesis of flavonoids, isoflavones, plant hormones, glutathione (GSH), soluble sugars, and other substances, as well as in ABC transporter and MAPK signaling pathways. Notably, the MT-treated group showed greater enrichment of DEGs in these pathways, suggesting that MT mitigates salt stress by modulating the expression of genes related to phytohormones and antioxidant capacity. Overall, our findings provide valuable insights into the molecular mechanisms underlying MT-mediated salt tolerance in alfalfa, with important implications for breeding salt-tolerant alfalfa and other crops.

3. Line 48, worldwide. [4]. Should be “worldwide [4].” More need to be fixed.

4. Line 72, the Introduction ended with “We hypothesized that melatonin combats salt stress by mediating the expression of genes associated with plant hormones and antioxidant capacity and conducted transcriptomic analyses aimed at revealing key genes and pathways of melatonin-mediated salt tolerance in alfalfa.” Should also include “Transition to the next section: Introduce the next section of the paper, such as the methodology, results, or discussion. Provide a brief overview of what will be covered in that section to guide the reader through the paper's structure. Conclude with a statement of significance: Emphasize the importance of the research and its potential implications for the field. Highlight why the study is relevant, timely, or innovative, and underscore the contribution it makes to advancing knowledge in the area.”

5. Line 88, how “10 µM MT” was selected? Give citations or explanations.

6. 3. Result Analysis should be “3. Results and Analysis”.

7. Line 154, Figure 1. Melatonin eliminates the inhibitory effect of salt stress on seed germination. Here, alleviates is better than eliminates.

8. Line 166, please revise “Under combined melatonin 166

and NaCl treatment, the accumulation of O2- and MDA decreased by 28.7% and 21.1%(Fig-167

ure 2 A-A), respectively, while the content of GSH, activity of SOD and POD increased by 168

54.7%, 43.1% and 2.5%(Figure 2 C-B), respectively. 169”

Figures should be cited in the order. Should be cited as Figure 2A, Figure 2B, or Figure 2C (no space between figure number and letter.

9. Line 180, “were shared by ZMNMT and ZMCK, ZMN and ZMNMT”—please correct.

10. Line 269, “(Such as CHS, E5.5.1.6, CYP73A, CYP75B1,” should be “(such as CHS, E5.5.1.6, CYP73A, CYP75B1,”.

11. The 5. Conclusion should be polished, one suggestion:

“In summary, our comprehensive physiochemical and transcriptomic analyses have elucidated the mechanisms underlying melatonin (MT)-mediated salt tolerance in alfalfa. MT supplementation enhances salt tolerance by promoting plant growth and bolstering antioxidant capacity. Additionally, MT treatment under salt stress conditions induces significant transcriptomic changes, particularly in genes associated with flavonoid biosynthesis, plant hormone signaling, and MAPK signaling pathways. Through weighted gene co-expression network analysis (WGCNA), potential hub genes linked to salt-responsive traits have been identified, suggesting their involvement in MT-regulated salt tolerance. These findings shed light on the molecular mechanisms underlying MT-mediated salt tolerance in alfalfa and offer valuable insights for breeding salt-tolerant varieties.”

12. Line 411, “Supplementary Materials:.”, please complete.

Comments on the Quality of English Language

Author Response

Thank you for the valuable suggestions provided by the reviewer. We fully agree with your comments and have made point-to-point revisions to the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Author Response

Thank you for the valuable suggestions provided by the reviewer. We fully agree with your comments and have made point-to-point revisions to the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Liu et al. offers a pivotal study on melatonin's role in enhancing alfalfa's tolerance to salt stress, a significant agricultural challenge. It explores this through comprehensive physiological, biochemical, and transcriptomic analyses, showing melatonin's efficacy in improving growth under salt stress by modulating key genes within flavonoid biosynthesis and antioxidant pathways. This suggests a promising avenue for developing salt-tolerant crops to mitigate soil salinization's impact.

The article is logically structured, moving seamlessly from introduction to conclusions, with a detailed experimental design covering control, salt stress, and melatonin treatments. The analysis spans germination rates, growth parameters, and oxidative stress markers, concluding with transcriptomic insights. The presented results articulate the role of DEGs in salt tolerance and melatonin response, supported by informative figures and tables. The discussion links findings to existing research, proposing mechanisms like flavonoid biosynthesis and MAPK signaling as pivotal to melatonin's effects.

Minor comments for enhancement include:

In the introduction section, consider providing a brief background on the importance of alfalfa as a forage crop and the challenges posed by soil salinity, to better contextualize the study's significance. Integrating a brief overview of current gaps directly related to this research could further justify the study's necessity. Explicitly stating the specific research gap and how this study addresses it would strengthen the introduction.

In the materials and methods section, provide more details on the experimental design, such as the number of replicates, the statistical analyses performed, and the criteria used for selecting the DEGs. Additionally, the section could benefit from additional details regarding the control setups and the rationale behind the choice of specific concentrations and experimental conditions.

Consider the flow and structure of the manuscript to ensure that it logically progresses from the introduction to the conclusion. In the results section, consider reorganizing some sections for better flow and clarity. For instance, the section on physiological and biochemical indicators could be presented before the transcriptomic analysis section.

The discussion effectively contextualizes the findings within the broader field of plant stress responses. Expanding on the potential underlying mechanisms influenced by melatonin would enrich this section. In the discussion section, some subsections could be merged or reorganized to avoid redundancy and improve coherence. Additionally, consider providing more insights into the potential practical applications of the findings, such as the implications for breeding salt-tolerant alfalfa varieties. Briefly acknowledge study limitations and suggest future research directions to demonstrate a critical understanding of the study's scope.

Overall, this manuscript contributes valuable insights into melatonin's potential in salt stress mitigation in alfalfa, with recommended revisions aimed at strengthening clarity, coherence, and impact.

Comments on the Quality of English Language

Review the manuscript for any typos, grammatical errors, or inconsistencies in formatting or style.

Author Response

Thank you very much to the reviewer for patiently reading our manuscript and providing some very useful suggestions.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The presented manuscript focuses on melatonin (MT) as a potential compound which may help alleviate the salt stress. The authors chose to conduct their experiments on alfalfa as one of the most important forage crop in the world. The topic is interesting and the results are worth publishing in Agriculture. In my opinion, however, the manuscript needs significant changes before publication. I also have some doubts about the methods (specified below). Moreover, by just googling Alfalfa, melatonin and salt stress, I found several studies conducted in the recent years, but these are not cited by the authors, e.g.

- Guo et al. (2023) Agronomy 13: 1727

- Jalili et al. (2023) Biologia 78: 961-970

The title generally reflects the content of the article, though the manuscript is not limited only to transcriptomics, so the authors may think about changing it.

The abstract states that the bud length was higher after the addition of MT (line 21), but this result was not statistically significant. Also, it should be specified that transcriptomics analysis was carried out in the seedlings roots (line 26).

Line 82: Why the authors used this specific salt concentration?

Lines 96-99: Specify if it was the content or activity measured in each case.

Line 102: How many root samples were used per biological replicate? Was it only from one seedling or a mixture of a few?

Lines 110-114: This description requires more details: What was the cut-off for low quality reads? High N rates – how high exactly? “concise length” – what does it mean?

Paragraph 2.7 – there are no results of qRT-PCR analysis. If the authors add them to the revised version of the manuscript, I still have some doubts: What were the criteria for the choice of genes? There is also no information about the reference genes used to normalize gene expression levels.

Line 150: The result obtained for the bud length was not statistically significant.

Lines 151-152: These last two sentences should be moved to the discussion.

Line 168: Figure 2 A-A à Figure 2 A-B.

Line 169: Figure 2 C-B à Figure 2 C-E.

Figures 2 and 3: There are different p-value levels in the caption but there are no corresponding asterisks in the figure itself.

Lines 208-212: ZMN and ZMNMT à ZMN vs ZMNMT, etc.

At some point, the results are mixed with the discussion, while these sections should be separated.

Figure 4: It is very small and has low quality. It is particularly difficult to read the text. I suggest to leave only A in the main text and make it bigger. B-D may be moved to the supplementary material.

Lines 338 as well as 392-399 need references.

Minor comments:

Line 82: MT abbreviation should be explained when first mentioned.

Line 84: placed à was placed

Line 135: differential genes à differentially expressed?

Line 140: Result Analysis à Results

Line 224: DEGs is à DEGs are

Line 231: This study obtained à This study identified/revealed

Line 260: write here that flavonoids may act as antioxidants

Line 268: should it not be like this?: we found that the number of DEGs related to (…) was higher, which indicates that melatonin…

Line 348: “amino acids and sugars”, but what? Metabolism?

Comments on the Quality of English Language

Minor changes needed (specified in the Comments and Suggestions for Authors)

Author Response

Thank you very much to the reviewer for patiently reading our manuscript and providing some very useful suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript needs more polishing for mistakes such as:

in author list: " Hao Sun 1,2,3, * Xiaoyan Zhu 1,2,3 and *".

Figure 1C and 1D figure legend.

Figure labels of Figure 1A, 1B are smaller than in Figure 1C and 1D.

Following examples of papers published in Agriculture, check if all supplementary figure and table titles should be listed in Supplementary Materials.

The format from Supplementary Materials to Conflicts of Interest, not all texts should be bold.

"and sequences with concise length (Discard sequences with length less than 20bp after removing the adapter and quality trimming). Then, mapped data (reads) for subsequent transcript assembly and expression calculation were obtained by comparing them with reference genomes [28]." Here should be "(discard....."

>>Response: Based on the phenotype data of alfalfa seedlings between different treatments, we detected that 10 μM exogenous MT could alleviate salt stress in alfalfa, which was consistent with Jalili, S.; Ehsanpour, A.; Morteza Javadirad, S. Biologia, 2023, 78, 961–970, line130.

Write in the manuscript.

Comments on the Quality of English Language

The manuscript needs more polishing.

Author Response

Thank you for the valuable suggestions provided by the reviewer. We fully agree with your comments and have made point-to-point revisions to the manuscript.

Author Response File: Author Response.pdf