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The authors have significantly improved the manuscript. It can now be accepted for publication.
The manuscript is significantly improved. I have included a few minor suggestions for your consideration (file attached). You are requested to address the reviewer's comments and my suggestions to revise the current version of the manuscript.
The requested changes were made. A few more points to note:
• Lines 70 and 88: italicize Arabidopsis thaliana
• Line 73: Italicize gene name
• Line 107: distinguish should be plural
• Line 112: a 1500 bp sequence upstream…
• Line 118: the degrees Celsius symbol (in 28º C) isn’t written like the other times you mentioned it correctly (e.g. 25 °C)
• Line 131: the -1 in µL-1 should be written in superscript
• Lines 151-152: units are missing
• Line 185: Gene structure “of"? Also, it’s gene structure you’re looking at, so it can’t be of the ARR-B proteins like you mentioned, but of the genes
• Line 255: sentence needs rewording
The requested changes were made.
• The required changes were made. If student’s t-test was used to look for significant differences in gene expression, this could be mentioned in the Methods section.
• In the abstract, you should talk about the genes that were significantly up-regulated in roots and/or leaves during salinity stress, rather than whether they were highly expressed in roots (line 30). If I were studying pepper plants, I would want to know at a glance (by reading your abstract), which would be the best genes for future functional validation studies on their roles in salinity stress. You mention CaARR-B3, -7, and -8 in the abstract, even though its expression is down-regulated under salinity stress in the root.
no comment
The reviewers have suggested major revisions for your manuscript. You are requested to address all the comments.
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The manuscript entitled with “Genome-wide identiûcation and expression analysis of CaARRs-B transcription factor gene family in pepper (Capsicum annuum L.) under salinity stress” by Alshegaihi et al provide some knowledge of pepper ARR-B family. The gene number, distribution, structure, and protein properties of CaARR genes were analyzed, and their expression patterns in response to salt stress were analyzed. This manuscript contributes to the knowledge of ARR-B gene family in pepper. However, it lacks novelty, and some further experiments are required. The major comments are listed as follows.
1. The language is not good, and there are many grammar mistakes. So, an extensive language processing is required.
2. The title is not accurate. “Genome-wide identification of CaARRs-B transcription factor gene family in pepper and their expression patterns under salinity stress” might be better.
3. The abstract, the language is not concise. For example, in lines 7-8, the sentence “phylogenetic relationships, gene structures, protein motif arrangement, and RT-qPCR expression” is repeat with the sentence in lines 13-14.
4. In the introduction, some simple introductions of CKs and ARR-B were included. the roles of CKs and ARRs in the stress responses, especially in salt stress responses are lacked. And also, are there some reports about the functions of pepper ARR-Bs? Because the authors said that “In pepper, only a few members of the ARRs-B family have been reported and characterized. in the abstract section.
5. Materials and Methods: The ARR-Bs in tomato have been identified (Wang J, Xia J, Song Q, Liao X, Gao Y, Zheng F, Yang C. Genome-wide identification, genomic organization and expression profiles of SlARR-B gene family in tomato. J Appl Genet. 2020, 61(3):391-404. doi: 10.1007/s13353-020-00565-5.). The authors should use tomato ARR-Bs as queries in blast and other bioinformatic analysis. Page 8 line 97, what is “GSDS”?
6. Results section:
(1) The subtitles should be corrected.
(2) In 3.4 section, there are many transcript isoforms for each CaARR, however, whether these transcripts really exist? Some RT-PCR experiments should be performed to confirm these transcripts.
(3) The tissue expression patterns of CaARRs should be performed.
(4) Why 12h and 24h of salt treatment were choosed?
7. Discussion section:
The authors used two paragraphs to discuss the roles of segmental duplication events in the CaARR gene duplication however, in the results section, no corresponding analysis (such as KaKs calculation) was performed.
(2) Why and how CaARRs are involved in salt stress repsonses, it is not discussed.
8. The conclusion section:
In Line 299, the authors said that “shed new light on CaARRs-B transcription factors’ role in secondary metabolism”. However, no result was found about the secondary metabolism.
The authors performed a genome-wide analysis of the ARR-B transcription factor family in Capsicum annuum and investigated the expression profiles of these genes under salinity stress. This research is a good foundation that can be used for further research involving functional validation studies. Therefore, I recommend that the manuscript be accepted for publication after completion of major revisions.
The following changes can be made to improve the quality of the grammar used throughout the manuscript:
• Lines 15-17: needs restructuring
• Line 23: replace with “functional divergence”
• Lines 28-30: needs restructuring
• Lines 32-34: could be worded better
• Lines 74-76: needs restructuring
• Line 80: should it be “The Arabidopsis thaliana sequences…”
• Line 99: The citation of Marchler-Bauer et al 2015 is for the NCBI CDD, not the GSDS database that was discussed in that line.
• All the instances of “cis” in ”cis-element” needs to be checked for italicization
• Line 104: sentence needs rewording
• Line 111: “28º C following” needs checking
• Lines 123-125: recheck
• Lines 143-144: The units for some of the quantities (e.g. MW) are missing.
• Lines 151-153: This line looks like it should be put in the methods section, especially since there was no mention of these programs in the methods section.
• Line 165: replace the phrase “take place”
• Line 173: “… and the lowest…”
• Line 212: I don’t see any highlighting of significant differences in Figure 5
• Lines 232-235: needs restructuring
• Line 249: The citation of Marchler-Bauer et al 2015 is for the NCBI CDD, not SMART
• Lines 247-251: These belong in the methods section and not discussion
• Line 264: “family genes”
• Line 278: “Despite this, ARR-B expression patterns in different tissues have rarely been detected in Pepper.” Didn’t you detect expression of these genes? This sentence needs clarifying.
• Line 284: “downregulated on in”
• All instances of the words downregulated and upregulated should be hyphenated (down-regulated, up-regulated)
• Line 297: “… have functionally…”
• Throughout the manuscript, the gene family is referred to as the ARRs-B family, while in the discussion, it is referred to as the ARR-Bs. The latter seems more grammatically correct.
The methods look experimentally sound and all underlying datasets have been provided. The following points can be used to improve the manuscript.
• In the manuscript, GAPDH is listed as the housekeeping gene. Actin is listed as the housekeeping gene in the Supplementary file. Please explain this discrepancy.
• The primers for the housekeeping gene are missing from Table 1.
• Was there z-score normalization of the heatmap values? Is so, this can be included in the figure legend.
• The authors referred to the expression values as fold changes, although the data calculations in Supplementary File 1 only show calculations for relative expression values. The findings could be made more robust by calculating the fold change of the 12H sample expression relative to that of the control (and similarly, for the 24H samples). An appropriate statistical test could be used to determine whether there are statistically significant differences in gene expression. This would allow the author’s claims of significant expression changes (Line 212) to be more scientifically sound.
• Lines 253-254: “… suggesting the family consists of segmental repetitions”. How does the location of the genes on 7 chromosomes suggest this? If this information was presented in previous literature, then it needs to be cited. Otherwise, I find it hard to accept this statement.
• Lines 30-32: “In particular, the expression of CaARR3, CaARR5, CaARR6, CaARR7, and CaARR8 showed significant expression levels in roots.” The title of this manuscript deals specifically with the roles of ARR-B genes during salinity stress. If a researcher wanted to conduct further research on this topic, they would want to see in the abstract at a glance which of these genes were the most likely to be involved in the pepper salinity stress response, yet no mention was made of such genes in the abstract or conclusion. If the suggested fold change calculations (see point 1 of this section) were performed, these results could be presented as part of the abstract.
no comment
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