Genome-Wide Identification and Expression Analysis of BrBASS Genes in Brassica rapa Reveals Their Potential Roles in Abiotic Stress Tolerance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors do an analysis of the BASS genes in Brassica rapa to elucidate their function in stress response.

The manuscript has some problems with gene naming. In a lot of places the genes are used with their numbers, but without species. As the genes have different numbers depending on the species, a conclusion drawn from e.g BASS2 is not valid in another species, as the orthologous gene is BASS3.
The naming scheme for miRNAs is not consistent throughout the text.
Explanations of gene names after abbreviations should not be all capitals.

Some paragraphs in the method section are hard to understand. Especially 2.5 and 2.6. In addition, why did you use two different versions of Excel?

There is no explanation to Figure 1, how the families have been defined. Figure 1B is not a representation of gene expression.

From Figure 2 it is also not obvious why it should support the grouping from Figure 1.

In paragraph 3.3 The description of the domains is not comprehensible and the number of motifs identified is misleading. Two of the proteins have only 9 motifs, that the other have 10 motifs might not be the truth, as the analysis has been limited to 10 motifs.

The paragraph describing the results for figure 3 is mentioning the lack of promoter elements for 'endosperm expression', but there is no information about the lack of elements for 'wound-responsive'.

In paragraph 3.6 the description of the expression changes is not consistent with the results in Figures 5, 6 and 7.

Figure 9: There are two genes from the same gene family in A and B, with different names: in A the gene is called ANTR1, which is the same as PHT4;1 and in B the gene is called PHT4;4, which is the same as ANTR2. You should use the same naming in both subfigures and I would suggest to use ANTR1 and ANTR2.

Table 3: The headings of the columns do not make sense.

Lines 47-50: The text is not consistent between wording and species and genus descriptions.

Comments on the Quality of English Language

Some editing is needed. Several sentences are not complete or structurally wrong.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Genome-wide identification and expression analysis of BrBASS genes in Brassica rapa reveals their potential roles in abiotic stress tolerance” aims to identify and characterize the Brassica rapa BASS gene family through bioinformatic, in silico and transcriptional analyses. The authors identified eight BrBASS family members by BLASTP using Arabidopsis BASS genes as queries, and then explored their molecular features through a series of analyses. In addition, the authors performed RT-qPCR analysis to examine the expression patterns of 7 selected candidate genes under various environmental stimuli. Based on the results, they concluded that BrBASS2, BrBASS4, and BrBASS7 genes were potential candidates for future breeding abiotic stress tolerant cultivars. Overall, the design and analysis of this study are correct, and the results are exhaustive. However, some results are required to re-examine, and several conclusions are overreached. Below, I outlined some of my major concerns that need to be addressed before next submission.

Generally, the language of this manuscript is required to be extensively examined.  

INTRODUCTION:

The authors provided comprehensive information to introduce the research history of BASS family in plants, which is helpful for readers understanding their biological importance. They also exemplified several previous transgenic studies of plant BASS genes in response to different abiotic stresses, enhancing the significance of current study. I suggest slightly expanding the background regarding the severe damages and agricultural losses of Chinese cabbages under abiotic and biotic stress, to emphasize the cruciality to understand the molecular mechanism of BASS gene family to environmental stimuli.

M&M:

Line 88, “aa” amino acid

Line 110, 2000 bp

Line 116, the authors re-analyzed the tissue-specific expression pattern of BrBASS members using publicly available data. The original article that created the dataset should be cited.

Line 123-124, the growth condition should be described in details, such as temperature, light intensity, humidity, etc.

Line 127, PEG6000 (W/V)

Line 127, the seedlings used for cold treatment were grown hydroponically as well?

The program used for phosphorylation site prediction is missing.

Statistical analysis should be detailed.

RESULTS:

Line 161-162, I don’t think these 8 BrBASS members are named based on Physical location. If so, BrBASS6 should be BrBASS2, and so on.

Section 3.2, the collinear pairs should be specified.

Figure 1b, the values above or below the colored bars need to be specified.

Figure 2c, I am interested to know where the transmembrane fragments and conserved Na+ binding sites located in each BrBASS protein.

Figure 2d and Table 2 are redundant.

Section 3.5, it is hard to speculate the gene function based on its tissue specific expression pattern, such as BrBASS1, 2 and 7.

Section 3.6, the authors should explain the absence of BrBASS1 in the RT-qPCR analysis.

Figure 5, statistical analysis of BrBASS4 is missing. The 2 hr time point (indicated in the legend) is missing as well.

Figure 8b, the signature helices of BASS could be indicated in each 3D model.

Section 3.8, it seems the authors used the Arabidopsis BASS proteins as queries, to explore the interactome by STRING database. Therefore, their findings are irrelevant to present study.

Figure 10, what is the meaning of y-axis?

DISCUSSION:

The third paragraph, discussing the chloroplast is important for abiotic stress response, is irrelevant to present study.

The ABA-responsive elements are present in the promoter region of all BrBASS genes. It is worth to detect the expression changes of BrBASSs under ABA treatment. The results would be helpful to determine whether BrBASSs participate in ABA-dependent signaling pathway under abiotic stress treatments.

DELLA, not DELA.

“Salt stress may be alleviated by BrBASS2.” Unless the function has been examined by molecular genetics study, such as overexpression and knockout mutant, it is unable to speculate gene function by transcriptional analysis.

CONCLUSION:

BrGeBP7?

Comments on the Quality of English Language

 Extensive editing of English language required

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have clearly addressed the most of my concerns, and the quality of the manuscript has been improved significantly. However, I have a few additional suggestions to improve the readability of the manuscript.

1.      Response 16 should be integrated into the Results or Discussion.

2.      Response 17.1, I suggest the authors re-examining the statistical results of BrBASS4.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear reviewers:

Thank you for your valuable comments on our manuscript once again. We appreciate your feedback and will respond to your questions accordingly.

For ease of discussion, we will first reprint your comments in italics and then present our response to them.

Point 1：

Response 16 should be integrated into the Results or Discussion.

Response 1：

We provide a detailed explanation of BrBASS1 in the Results section regarding its consistently low and stable expression levels and an explanation of its potential functional similarity to the Arabidopsis homologue BASS5, which plays an important role in methionine-derived glucosinolate biosynthesis. “In addition to these genes, we also analyzed the expression of BrBASS1 under the same stress conditions. The results showed that the expression level of BrBASS1 remained consistently low and did not exhibit significant changes across all the time points studied. This suggests that BrBASS1 may not play a significant regulatory role under osmotic, cold, or salt stress conditions. Furthermore, its homologous gene in A. thaliana, BASS5, has not been extensively studied under stress conditions. Therefore, BrBASS1 was not included in the detailed analysis and discussion of stress-responsive genes.” Line265-272. we hope that our explanations are helpful in answering your questions.

Point 2：

Response 17.1, I suggest the authors re-examining the statistical results of BrBASS4.

Response 2：

Thank you for your valuable feedback on our study. In response to your suggestion regarding the statistical results of BrBASS4, we have re-examined the expression data by performing additional RT-qPCR experiments. We have re-analyzed the data and recalculated the statistical significance using t-tests.

The updated Figure 5, now included in the manuscript, reflects these new results. The expression levels and significance markers for BrBASS4 have been updated accordingly.

We appreciate your constructive comments, which have helped us improve the accuracy and clarity of our findings.

Best wishes

Ruolan Wang

Author Response File: Author Response.pdf