Identification of Glucose-6-Phosphate Dehydrogenase Family Members Associated with Cold Stress in Pepper (Capsicum annuum L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,
I consider that your article presents relevant information and constitutes an important scientific contribution. However, in some sections, the article lacks consistency and clarity. Attached is a file detailing this and providing suggestions for improvement.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study presents a scientifically and methodologically sound investigation into the G6PDH gene family in Capsicum annuum ('Zunla-1'), with a particular emphasis on the functional role of CaG6PDH2 in the cold stress response. Investigating G6PDH in Capsicum annuum is highly relevant, particularly in the context of cold stress tolerance. The identification of four G6PDH genes (CaG6PDH1–CaG6PDH4) from the 'Zunla-1' genome is consistent with gene family sizes in other dicot plants, and the use of sequence alignment, motif identification, gene structure, and collinearity analysis reflects good bioinformatics practice. Moreover, positioning the Capsicum G6PDHs among Solanaceae orthologs enhances evolutionary interpretation and supports the prediction of gene function.

 

However, with the following clarifications and full methodological details, the study could be suitable for publication:

1/ The number of biological replicates, statistical methods, or whether differences were statistically significant are important for scientific rigor.

2/ While expression and localization are shown, direct measurement of G6PDH enzymatic activity in silenced vs. control tissues would strengthen functional claims.

3/ VIGS can have off-target effects; confirmation of gene knockdown specificity (e.g., via qRT-PCR of off-target genes or rescue experiments) would improve confidence in the conclusions.

4/ It should be acknowledged that VIGS provides only transient gene silencing and may not fully replicate stable gene knockout phenotypes.

5/ The localization of CaG6PDH2 in chloroplasts is essential; however, it should ideally be validated using fluorescent fusion proteins (such as GFP assays in Nicotiana).

6/ The dynamics of gene expression or ROS over time under cold stress would add depth to the characterization of stress responses.

7/ The title of section 3.1 appears to be incorrect (“MeDIRs in Cassava Genome”). It should accurately reflect the focus on G6PDH genes in the Capsicum annuum genome.

8/ Species names should be italicized consistently. Minor typographical errors ("gnes" in section 2.2 of Materials and Methods) should be corrected. The manuscript contains numerous grammatical and stylistic issues that hinder readability. Consider professional editing for fluency and consistency.

Comments on the Quality of English Language

Species names should be italicized consistently. Minor typographical errors ("gnes" in section 2.2 of Materials and Methods) should be corrected. The manuscript contains numerous grammatical and stylistic issues that hinder readability. Consider professional editing for fluency and consistency.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,
Thank you for taking the time to address all my queries and for your efforts to incorporate my recommendations. However, the following concern persists:
In their response to the reviewers, the authors state, 'To investigate the role of the CaG6PDH2 gene, we only conducted cold treatment in this study. Therefore, drought, heat and salt stress were not considered.' However, why did the authors include the following paragraph in the methodology in 2.11 Pepper Materials and Stress Treatment? 'Heat stress was applied at 39 °C, with leaf samples collected at 3, 6, 12 and 24 hours, respectively.' Drought stress was induced by withholding water and leaf samples were collected at 1, 2, 3 and 4 days (d) respectively. Salt stress was induced by applying 250 mmol/L sodium chloride, with leaf samples collected at 6, 12, 24 and 48 hours, respectively. Untreated seedlings served as the control group (CK, 0 h). Each treatment included three biological replicates, and leaf samples were immediately frozen in liquid nitrogen and stored at -80 °C..." '

If the treatments were not included in the study, why are they declared in the methodology?

I apologise if I have misunderstood something.

Author Response

Comments 1: In their response to the reviewers, the authors state, 'To investigate the role of the CaG6PDH2 gene, we only conducted cold treatment in this study. Therefore, drought, heat and salt stress were not considered.' However, why did the authors include the following paragraph in the methodology in 2.11 Pepper Materials and Stress Treatment? 'Heat stress was applied at 39 °C, with leaf samples collected at 3, 6, 12 and 24 hours, respectively.' Drought stress was induced by withholding water and leaf samples were collected at 1, 2, 3 and 4 days (d) respectively. Salt stress was induced by applying 250 mmol/L sodium chloride, with leaf samples collected at 6, 12, 24 and 48 hours, respectively. Untreated seedlings served as the control group (CK, 0 h). Each treatment included three biological replicates, and leaf samples were immediately frozen in liquid nitrogen and stored at -80 °C..." '

If the treatments were not included in the study, why are they declared in the methodology?

I apologise if I have misunderstood something.

Response 1: Thank you very much for your comment. The manuscript is divided into three main sections. The first section focuses on bioinformatics analysis. The second section discusses the expression of the G6PDH gene in various tissues, as well as its expression profile in response to plant hormones and stress. In this section, we first analyzed the transcriptome data and then validated its accuracy using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Consequently, the cold, heat, drought, and salt treatments applied to pepper, as mentioned earlier, were conducted for quantitative analysis, establishing a foundation for exploring the function of the G6PDH gene under stress conditions. This section is located in the second paragraph of section 2.11, Pepper Materials and Stress Treatment. The final section employs Virus-Induced Gene Silencing (VIGS) technology to investigate the function of the CaG6PDH2 gene under cold stress, necessitating the implementation of various cold treatments. This section is located in the third paragraph of section 2.11.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have thoroughly addressed all the comments raised in a clear and detailed manner. Their responses demonstrate careful consideration of the suggestions provided, making the manuscript suitable for publication.

Author Response

Comments 1: The authors have thoroughly addressed all the comments raised in a clear and detailed manner. Their responses demonstrate careful consideration of the suggestions provided, making the manuscript suitable for publication.

Response 1: Thank you very much for your comment.