Genome-Wide Analyses Reveal the Roles of CCT Genes as Regulators of Abiotic Stress Responses in Citrullus lanatus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

My recommendations for the manuscript entitled ‘Genome-wide analyses highlight the essential roles of CCT 2 genes as regulators of Citrullus lanatus responses to prolonged 3 light exposure and abiotic stressors’ are mentioned below. The authors need to critically revise it before it is accepted for publication.  

Ø  The title of the paper is relatively long and needs to be shortened/modified as: ‘Genome-wide analyses reveal the roles of CCT 2 genes as regulators of abiotic stress responses in Citrullus lanatus’.

Ø  The authors need to ensure that Introduction section is laid out in a manner that adequately captures and presents the need and rationale for the present study. It should start with the area and production statistics of this crop in China, how it is impacted in terms of yield and fruit quality by various abiotic stresses in general and the stresses imposed in this study in particular.

Ø  The Introduction section should also adequately highlight how salt and other stresses impact the watermelon plants at the cellular, tissue and whole plant levels. Then, authors should describe the physio-biochemical and molecular underpinnings for adapting to these stresses.

Ø  In the Materials and Methods section, ‘Site Description and Experimental Material’ and ‘Stress Treatments’ should be presented separately. It is entirely unclear how salt and other stress treatments were imposed. Therefore, this ambiguity should be overcome and various procedures should be presented in some detail. The agro-climatic conditions such as temperature and RH during the course of experiment should be properly described. Other subsections (2.1 to 2.4) should be presented thereafter.

Ø  The statement that ‘Watermelon variety 97,103 plant materials were utilized for analyses of CCT gene expression profiles in different tissues conducted at the Institute of Vegetables at Ningbo Academy of Agricultural Sciences’ is vague and needs to be looked into. It is grossly unclear whether this is the total number of experimental plants or the number of samples. Moreover, the experiments were conducted on any specific watermelon variety? Provide the details of variety.

Ø  The manuscript is replete with grammar and other errors. Careful English editing is needed to improve the quality. I have noticed several vague and ambiguous sentences including those in lines 74-77 [Watermelon (Citrullus lanatus) is an economically important melon crop throughout the globe, and China is the worldwide leader in terms of both watermelon output and  cultivation area, with watermelons being a popular fruit that is widely consumed in China], 81-82 (No similarly systematic analyses of the watermelon CCT gene family have yet been conducted, however), 289-291 (To more fully understand the processes that govern the expression of ClCCT genes and their associated functions, 70 total predicted cis-acting elements were located within the 1.5 kb regions upstream of the translation start sites for these ClCCT family members using the PlantCARE database). These are some examples only. Authors are advised to carefully look into whole manuscript to address such comments.

Ø  Authors have not presented any basic morphological and physiological changes caused by the stresses imposed in the present study. This is, however, required to make this study more attractive and appealing to the prospective readers.

Ø  Authors need to place more emphasis to adequately highlight what this experiment produced in terms of groundbreaking knowledge that did not exist before. Therefore, changes are required in Discussion section to highlight the novelties of the present study.

Ø  The Abstract section should briefly and explicitly mention the names of stresses imposed in this experiment.

Ø  The Conclusions section is quite short, and needs to be elaborated further. Along with key findings, this section should also present the future course of action and further experiments required.

Ø  Under the head ‘Informed Consent Statement: Informed consent was obtained from all subjects involved in the study’ what were those subjects?.

Comments on the Quality of English Language

Must be improved, Careful Editing and Proofreading are required. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the manuscript “Genome-wide analyses highlight the essential roles of CCT genes as regulators of Citrullus lanatus responses to prolonged light exposure and abiotic stressors” effectively showed the importance of the CCT gene family in environmental adaptation and photoperiodic flowering. This article analyzed the physicochemical properties, duplication of CCT gene events, and abiotic or hormonal responsiveness

Some specific issues are as follows.

Abstract
(1) contains most of information like gene duplication, motifs, structural divergence and responsiveness in a limited space, focus on most critical information.

(2)The experimental evidence for the CCT gene’s role in flowering and stress response is only suggested? Briefly mention the experimental outcomes.

(3) For details of clear finding add, how many genes were analyzed, levels of expressions or responsiveness

(4) Few terms like segmental duplication, functional divergence, required brief clarification.

Introduction
(1) In introduction a concise information of CCT genes and their significance are lacking?? discuss their roles in flowering and stress responses.

(2) Some terms in L43, B-box domains, L44-REC domain  L 83- collinearity are need explanation
(3) Add sentences from general findings in other species to focus on water melon to bridge the  research gap and  objectives
(4) The role of CCT genes are repetitive,  avoid repetition and add new complementary information.
(5) Add information that indicate what makes this study novel and significant, potential outcomes from analyzes of watermelon CCT genes for future crop improvement.

M&M
(1) mention the description of  transcriptomic data and treatment sources avoid repeated data like  listing of treatments..
(2) Data bases used in this research study such as LocTree3 and WoLF PSORT why were these tools chosen for subcellular localization??
(3) State the technical replicates for qPCR analysis are not mentioned.

(4) limited information about descriptions of how phylogenetic tree results, genes structure visualization were interpreted, add information regarding criteria for analysis or threshold values.

(5) Sample collections for time point during prolong daylight are fine however, the right rationale for this sampling is missing.

(6) Statistical analysis for expression profiling important for validating the differences in genes expression under different condition are missing…

(7) Methods for clustering or colour coding are not clear.

(8) Mention the software or tools used for heatmap construction.

 Results
(1) some data mentioned repeatedly such as treatments and transcriptomic data…..revise for clarity.

(2)  The tools and data bases like LocTree3 and WoLF PSORT were chosen for subcellular localization, mention brief description why these software and tools were selected??

(3) The methods do not mention how statistical analyses, were performed for expression profiling, which is crucial for validating differences in gene expression under different conditions. Describe the statistical methods used for analysis of gene expression data.

(4) Give a rationale for the chosen timing and duration of sampling under stress and daylight conditions and relate this to biological relevance or previous findings. The exact rationale for this sampling schedule is missing.

Discussion
(1) in Discussion describing gene responses to stress and their promoter elements are repetitive, (L479-481) the discussion on ClCCT28’s responsiveness to many stress under salt conditions …… need clarity.

(2) Expand discussion on, how ClCCT16 and ClCCT1 may effect the flowering or stress resilience under different environmental conditions.

(3)  Specific gene response to the molecular pathways is missing, the subsequent effects of these abiotic stress responses are not discussed.

Figures and Tables
(1) Ensure that all figures and tables cited in the discussion such as Figure 7, Figure 9, Table S3
(2) connect the  all figure and table expressively with text and summarize their key findings concisely .

Conclusion

In conclusion the important findings such as identification of multifunctional genes like ClCCT28 or ClCCT4 similarly the insight into evolutionary relationships are missing.

 

 In conclusion the role of cis acting elements in stress responses or the diurnal expression patterns of certain ClCCT genes does not reflect the complete knowledge gained.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Title: Genome-wide Analyses Highlight the Essential Roles of CCT Genes as Regulators of Citrullus lanatus Responses to Prolonged Light Exposure and Abiotic Stressors

The manuscript aligns well with the journal's scope, presenting a comprehensive analysis of watermelon CCT genes, focusing on their physicochemical characteristics, gene duplication relationships, homology, and collinearity with other species. However, the following concerns and suggestions need to be addressed:

Keywords:
Consider adding 1–2 more relevant keywords, to improve discoverability.

 

Introduction:

1. Revise the statement in line 76: “Watermelons being a popular fruit” to reflect clarity and accuracy. Suggested revision: “Watermelons (Citrullus lanatus) are highly popular for their sweet and refreshing fruit, cultivated globally for consumption.”
2. Address the classification issue: “Watermelon, botanically classified as a vegetable.

 

Materials and Methods:

• Provide justification for selecting the watermelon variety 97,103 as plant materials. For example, explain if this variety was chosen due to its genetic diversity, stress tolerance, or significance in previous research. This adds depth and credibility to the methodology.

 

Results:

1. Identify and consolidate redundant figures that provide overlapping information. These could be moved to supplementary materials to streamline the results section and emphasize unique findings.
2. Ensure all figures and their legends are clear and concise, with sufficient explanation for independent interpretation.

Discussion:

1. Expand the discussion to focus more deeply on the relationship between CCT genes and their roles in abiotic stress responses.
2. Discuss the regulatory mechanisms of CCT genes in light exposure and stress tolerance, integrating insights from other crops, where applicable, to provide a broader context.
3. Emphasize the practical applications of your findings in breeding stress-tolerant watermelon varieties.

 

Conclusions:

• Enhance this section by succinctly summarizing the main findings, such as the key roles of CCT genes in abiotic stress responses and prolonged light exposure.
• Include implications for future research, emphasizing potential applications in crop improvement programs.

 

Formatting Notes:

1. Revise all instances of scientific names (e.g., Citrullus lanatus) to be written in italics for consistency and adherence to scientific standards.
2. Perform a final revision to ensure scientific names, gene names, and technical terminology are accurately formatted throughout the manuscript.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the manuscript “Genome-wide analyses highlight the essential roles of CCT genes as regulators of Citrullus lanatus responses to prolonged light exposure and abiotic stressors” modified the manuscript as suggested. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed the revisions comprehensively, and the manuscript now meets the required standards. It is suitable for acceptance and publication.