Genome-Wide Identification, Characterization, and Expression Profile of SWEETs Gene Family in Grapevine (Vitis vinifera L.)

Round 1

Reviewer 1 Report

The study entitled “Genome-wide Identification, Characterization, and Expression Profile of SWEETs Gene Family in Grapevine (Vitis vinifera L.)” reports a comprehensive bioinformatic characterization and analysis of the grapevine SWEET gene family leveraging the latest grapevine genomic data. Authors identified 18 members of the family and a description of genes structure, conserved domains, and phylogenetic relationships was conducted. Transcription profiles of SWEET genes showed tissue- and spatial-specificity which correlates with their respective clades.  Some members putatively involved in abiotic and biotic stress response have been identified, offering a fundamental reference for further functional studies. 

The study is quite well organized and results clearly presented. 

However, there is some issues which have to be addressed. 

In Material and Methods: 

Line 124. It is good practice to set the E-value <0.001, so the authors should explain why they used the E-value of 10e-2. 

Line 134. To reinforce data, it might be useful to use CLUSTAL for multialignment or perhaps explain why DNAMAN, less used in the literature, is chosen. 

Line 138. Results should be implemented with a maximum likelihood (ML) analysis and make a comparison between the two results to reinforce the phylogenetic output. 

Lines 191-192. Is there literature to be cited for these varieties? 

Line 195. For the inoculation approach a protocol must to be cited. 

Line 205. For the qRT-analysis the authors used the 2−ΔΔCt method, a very old and not precise approach. I strongly suggest to analyze data using the real amplification efficiency (Pfaffl , 2001; 

In Results section: 

Lines 231-234; 241-242; 263-267. These parts should be moved on Discussion section and authors should add a new dedicated paragraph.  

Line 349. It should be very useful to add pictures of leaves after Botrytis cinerea infection, in oder to appreciate the entity of disease. 

Author Response

Dear editor and reviewers,

Many thanks for spending time to review our work.

We are grateful to the Editor and anonymous reviewers for their valuable comments and suggestions which help to improve the quality of the paper. We have studied the reviewers’ comments carefully and have made modifications and corrections which we hope meet their approval.

In Material and Methods:

Q1:

Line 124. It is good practice to set the E-value <0.001, so the authors should explain why they used the E-value of 10e-2.

A1:

Thank you for your valuable comments. Regarding the choice of E-value, we used 10e-2 instead of a stricter 0.001 for the following reasons:

1.Basis in Previous Research: The setting of the E-value was based on several similar studies in the field:

Zhang, X., Zhang, L., Ji, M., Wu, Y., Zhang, S., Zhu, Y., Yao, J., Li, Z., Gao, H., & Wang, X. (2021). Genome-wide identification and expression analysis of the B-box transcription factor gene family in grapevine (Vitis vinifera L.). BMC Genomics, 22(221). https://doi.org/10.1186/s12864-021-07489-8.

This method was adopted from this grape gene family analysis article and has been cited in the revised manuscript.

Jiang, C., Zeng, S., Yang, J., & Wang, X. (2023). Genome-wide identification and expression profiling analysis of SWEET family genes involved in fruit development in plum (Prunus salicina Lindl). Genes (Basel), 14(9), 1679. https://doi.org/10.3390/genes14091679.

In this 2023 article on the SWEET family, the authors set the E-value < 1.0.

2.Balancing Discovery and Error Rates: Using an E-value of 10e-2 in our study helps to balance the rate of discovery and error, allowing us to capture more sequences with potential biological significance.

Q2:

Line 134. To reinforce data, it might be useful to use CLUSTAL for multialignment or perhaps explain why DNAMAN, less used in the literature, is chosen.

A1:

Thank you for your suggestions and attention to our choice of methods. We opted to use DNAMAN for the following reasons:

1.Basis in Previous Research: We referred to several similar studies within the field:

Zhang, X., Ma, J., Yang, S., Yao, W., Zhang, N., Hao, X., & Xu, W. (2023). Analysis of GATA transcription factors and their expression patterns under abiotic stress in grapevine (Vitis vinifera L.). BMC Plant Biology, 23, 611. https://doi.org/10.1186/s12870-023-04604-1.

This method was derived from this grape gene family analysis article and has been additionally cited in the revised manuscript.

Xie, H., Wang, D., Qin, Y., Ma, A., Fu, J., Qin, Y., Hu, G., & Zhao, J. (2019). Genome-wide identification and expression analysis of SWEET gene family in Litchi chinensis reveal the involvement of LcSWEET2a/3b in early seed development. BMC Plant Biology, 19(1), 499. https://doi.org/10.1186/s12870-019-2120-4.

In this article, DNAMAN was also used for multiple sequence alignment of SWEET proteins.

2.Verification with GLUSTAL Method: Additionally, we employed the GLUSTAL method and found that the results obtained were consistent with those from DNAMAN.

Q3:

Line 138. Results should be implemented with a maximum likelihood (ML) analysis and make a comparison between the two results to reinforce the phylogenetic output.

A3:

Thank you for your valuable suggestions. In this study, we have used the Neighbor-Joining (NJ) method to construct phylogenetic trees, primarily for the following reasons:

1.Robustness: The NJ method is robust and not overly sensitive to individual outliers. Even in the presence of some anomalies or errors in the data, it is unlikely to cause significant changes in the overall structure of the phylogenetic tree. In contrast, the Maximum Likelihood method might be affected by these outliers, potentially leading to larger errors.

2.Independence from Specific Evolutionary Models: Unlike some methods, the NJ method does not rely on specific assumptions about the sequence evolution model. Therefore, in cases where the evolutionary pattern is complex or unclear, it can still produce reasonable results.

3.Widespread Acceptance and Reliability: Based on previous research in this field, the NJ method has been widely accepted and employed in similar studies, where its results have been highly credible. This approach has also been used in the latest research on gene families, for example:

Deng, F., Wang, H., An, X., & Uwamungu, J. Y. (2024). A genome-wide analysis of the WUSCHEL-related homeobox transcription factor family reveals its differential expression patterns, response to drought stress, and localization in sweet cherry (Prunus avium L.). Horticulturae, 10(4), 370. https://doi.org/10.3390/horticulturae10040370

Q4:

Lines 191-192. Is there literature to be cited for these varieties?

A4:

Following your suggestions, we have added the following references regarding the varieties in the revised manuscript:

Wan, R., Hou, X., Wang, X., Qu, J., Singer, S. D., Wang, Y., & Wang, X. (2015). Resistance evaluation of Chinese wild Vitis genotypes against Botrytis cinerea and different responses of resistant and susceptible hosts to the infection. Frontiers in Plant Science, 6, 854. https://doi.org/10.3389/fpls.2015.00854

Gabler, F. M., Smilanick, J. L., Mansour, M., Ramming, D. W., & Mackey, B. E. (2003). Correlations of morphological, anatomical, and chemical features of grape berries with resistance to Botrytis cinerea. Phytopathology, 93(10), 1263-1273. https://doi.org/10.1094/PHYTO.2003.93.10.1263

Rahman, M. U., Hanif, M., Wan, R., Hou, X., Ahmad, B., & Wang, X. (2018). Screening Vitis genotypes for responses to Botrytis cinerea and evaluation of antioxidant enzymes, reactive oxygen species, and jasmonic acid in resistant and susceptible hosts. Molecules, 24(1), 5. https://doi.org/10.3390/molecules24010005

Q5:

Line 195. For the inoculation approach a protocol must to be cited.

A5:

We apologize for not citing the inoculation protocol used in detail in the original manuscript, which was an oversight on our part. We have now added the specific reference to the inoculation protocol used in the methods section. The inoculation method described in the following publication was referred to:

Wan, R., Hou, X., Wang, X., Qu, J., Singer, S. D., Wang, Y., & Wang, X. (2015). Resistance evaluation of Chinese wild Vitis genotypes against Botrytis cinerea and different responses of resistant and susceptible hosts to the infection. Frontiers in Plant Science,6, 854. https://doi.org/10.3389/fpls.2015.00854

Q6:

Line 205. For the qRT-analysis the authors used the 2^−ΔΔCt method, a very old and not precise approach. I strongly suggest to analyze data using the real amplification efficiency (Pfaffl , 2001;

A6:

Thank you for your concerns and suggestions regarding the qRT-PCR data analysis method we used. We understand the reservations about the 2^−ΔΔCt method, as you mentioned, this method may not be as precise as using the actual amplification efficiency method by Pfaffl (Pfaffl, 2001). Our decision to use this classic method was based on the following reasons:

1. Reliability of Experience: Although the 2^−ΔΔCt method has been around for many years, it is still widely used in research and has been validated across various fields. Its popularity and reliability make it an effective tool for assessing relative gene expression differences.

2.Continued Use in Recent Literature: Many recent publications still use this method for qRT analysis, for example,

Gong, Q., Wang, Y., He, L., Huang, F., Zhang, D., Wang, Y., Wei, X., Han, M., Deng, H., Luo, L., Cui, F., Hong, Y., & Liu, Y. (2023). Molecular basis of methyl-salicylate-mediated plant airborne defence. Nature, 622(7981), 139-148. https://doi.org/10.1038/s41586-023-06533-3

3. Appropriateness for This Study: In this study, due to factors such as experimental design and the number of samples, we believe that the 2^−ΔΔCt method meets our basic needs for result precision and reliability.

In Results section:

Q7：

Lines 231-234; 241-242; 263-267. These parts should be moved on Discussion section and authors should add a new dedicated paragraph.

A7:

Based on your suggestions, we have moved these contents to the Discussion section. Additionally, we have split the second paragraph of the Discussion section into two paragraphs in the revised manuscript.

Q8：

Line 349. It should be very useful to add pictures of leaves after Botrytis cinerea infection, in oder to appreciate the entity of disease.

A8：

Based on your suggestion, we have added images of leaves infected with Botrytis cinerea to the Supplementary File (Figure S1). These images, taken at different time points, visually illustrate the progression and severity of the disease.

Reviewer 2 Report

The manuscript characterized the SWEET gene family in grape vine (Vitis vinifera) species using bioinformatic methods and tools. Retrieving and using the available species genome along with genes annotations, the study identified the members of the SWEET family in the grape genome, assessed their phylogenetic relationships, predicted chromosomal localization, projected the exon-intron structures, and motif composition. Similarly, the authors conducted a transcriptome analysis using a set of selected grapevine species and cultivars along with transcriptome profiles available in datasets to assess the expression profile of the SWEET family at various fruit developmental stages, and under abiotic stress conditions. The study is well written providing a timely and thorough bioinformatics analyses based on available databases of grapevine genome and transcriptome profiles, by selecting and analyzing essential cultivars. Therefore, the authors are encouraged to clearly illustrate the study methodology by emphasizing the power of bioinformatic analysis, and to clearly illustrate the experiments performed. Thus, the following issues should be elaborated and clarified.

1.       In the Abstract ln 21-24 “Furthermore, …. infection” should be moved to ln 28 before “Expression profiling…”.

2.       Ln 28-29 “Expression profiling demonstrated strong tissue-specificity and temporal-spatial specificity of VvSWEETs, correlated with their respective clades” should be elaborated in the Results section to depict the tissue-specificity as genes’ expression in various tissues and time are not depicted in the text or Figures.

3.       The Materials and Methods sections should be rephrased to clearly depict how the bioinformatic analyses, as the only methods employed by the authors, were carried out. Thus, the subsections2.5 and 2.6 titles “Expression patterns in response to ….” is rather confusing/undervalued and should be rephrased accordingly. The subtitle could be “Analysis of expression profiles in….”. And the text followed should clearly depict the tissues, time and cultivars used in each dataset of transcriptomics. These can be depicted in a supplementary Table as these details can be found in the referenced articles of the datasets.  Moreover, it is suggested to comment on the cultivars used in the transcriptomic datasets as there are significant differences (e.g. resistant/sensitive to drought, cold or salt, etc.), highlighting the importance of the study to providing new knowledge on the role of the SWEET family genes in response to abiotic/biotic stress management in grapevine. The selection of the datasets of such cultivars is the novelty of the study and needs to be highlighted in the text. It is suggested to illustrate this in subsection 3.5 and 3.6 accordingly, demonstrating the impact of the study, and highlighting the use and value of information gained through these scrupulous analyses performed.

4.       Subtitle 2.7 could be rephrased to “Expression profiles to biotic stress” to indicate and encompass a different section of experiments and analyses than the above. Then the description of the plant material used could be illustrated at the beginning of the paragraph as follows: Eight grapevine cultivars were selected for resistance/susceptibility to Botrytis cinerea from the grapevine germplasm resource vineyard of Northwest A&F University, Yangling, Shaanxi, China (34°20′N, 108°24′E).  Specifically, young leaves of four resistant varieties ('Red Globe', 'Gebixinxiu', 'Thompson Seedless', 'JingXiangyu') and four susceptible varieties ('Shangyou', 'Beihong', 'Beimei', 'Gold Finger '), grown under natural environmental conditions were inoculated with Botrytis cinerea. Leaves samples were collected at 0-, 24-, and 48-hours post-inoculation. All samples were rapidly frozen in liquid nitrogen and stored at -80°C.”

5.        Ln 198-206, the reference gene used (i.e. ACTIN) in the RNA expression studies should be depicted here.

6.       Figure legend 6 “Expression of the date VvSWEET” the term “date” refers to the berry, that is the fruit unit composing the grape? Then it should be corrected.

7.       In the Discussion section ln 428-433 “wheat has the highest number of SWEET members” should be elaborated to indicate that cultivated wheat is either allotetraploid (AABB) for durum wheat (Triticum turgidum ssp. durum), or allohexaploid (AABBDD) for bread wheat (Triticum aestivum), thus justifying the high number of SWEET genes identified in a species with multiple genomes.

8.       In ln 430 “loquat” should be followed by the species name.

9.       Given the scrupulous bioinformatics analyses conducted in this study, I would suggest presenting a Table to depict the species assessed/compared and the number of SWEET family genes identified in each species, including the grapevine, with the relevant references.

Minor english editing errors.

Author Response

Dear editor and reviewers,

Many thanks for spending time to review our work.

We are grateful to the Editor and anonymous reviewers for their valuable comments and suggestions which help to improve the quality of the paper. We have studied the reviewers’ comments carefully and have made modifications and corrections which we hope meet their approval.

Q1:

In the Abstract ln 21-24 “Furthermore, …. infection” should be moved to ln 28 before “Expression profiling…

A1:

Thank you for your suggestion. Based on your advice, we have adjusted the placement of this section in the revised manuscript's abstract.

Q2:

Ln 28-29“Expression profiling demonstrated strong tissue-specificity and temporal-spatial specificity of VvSWEETs, correlated with their respective clades” should be elaborated in the Results section to depict the tissue-specificity as genes expression in various tissues and time are not depicted in the text or Figures

A2:

Thank you for your suggestion. For the figures describing the expression of SWEET in various tissues at different developmental stages, please refer specifically to "Figure 4 VvSWEET expression profiles in various tissues at different developmental stages." This figure includes 54 different developmental stage organs/tissues, and for a detailed description of the results, please see "3.5 Expression Analysis of Grape SWEET Genes in Different Tissues."

Q3:

The Materials and Methods sections should be rephrased to clearly depict how the bioinformatic analyses, as the only methods employed by the authors, were carried out. Thus, the subsections2.5 and 2.6 titles “Expression patterns in response to ….” is rather confusing/undervalued and should be rephrased accordingly. The subtitle could be “Analysis of expression profiles in….”. And the text followed should clearly depict the tissues, time and cultivars used in each dataset of transcriptomics. These can be depicted in a supplementary Table as these details can be found in the referenced articles of the datasets.  Moreover, it is suggested to comment on the cultivars used in the transcriptomic datasets as there are significant differences (e.g. resistant/sensitive to drought, cold or salt, etc.), highlighting the importance of the study to providing new knowledge on the role of the SWEET family genes in response to abiotic/biotic stress management in grapevine. The selection of the datasets of such cultivars is the novelty of the study and needs to be highlighted in the text. It is suggested to illustrate this in subsection 3.5 and 3.6 accordingly, demonstrating the impact of the study, and highlighting the use and value of information gained through these scrupulous analyses performed.

A3:

Thank you very much for reviewing our paper and for your detailed suggestions.

In the revised manuscript, we have changed the titles to "2.5. Analysis of Expression Profiles in Various Organs and Different Berry Developmental Stages" and "2.6. Analysis of Expression Profiles in Different Abiotic Stress Conditions" to accurately reflect the methods used. We have added supplementary Tables 1 and 2, which provide detailed descriptions of the tissues, timing, and cultivars used in each transcriptome dataset. Additionally, the cultivars used in the transcriptome datasets are further described in the corresponding Section 3.7.

Q4:

Subtitle 2.7 could be rephrased to “Expression profiles to biotic stress” to indicate and encompass a different section of experiments and analyses than the above. Then the description of the plant material used could be illustrated at the beginning of the paragraph as follows: Eight grapevine cultivars were selected for resistance/susceptibility to Botrytis cinerea from the grapevine germplasm resource vineyard of Northwest A&F University, Yangling, Shaanxi, China (34°20′N, 108°24′E).  Specifically, young leaves of four resistant varieties ('Red Globe', 'Gebixinxiu', 'Thompson Seedless', 'JingXiangyu') and four susceptible varieties ('Shangyou', 'Beihong', 'Beimei', 'Gold Finger '), grown under natural environmental conditions were inoculated with Botrytis cinerea. Leaves samples were collected at 0-, 24-, and 48-hours post-inoculation. All samples were rapidly frozen in liquid nitrogen and stored at -80°C.”

A4:

Thank you to the reviewer for the detailed guidance on this matter. Based on your suggestion, we have revised the title and content of Section 2.7.

Q5:

Ln 198-206, the reference gene used (i.e. ACTIN) in the RNA expression studies should be depicted here.

A5：

Thank you to the reviewer for your attention to this detail. Following your suggestion, we have made the necessary additions, as detailed in Section 2.8 "...with the grapevine ACTIN1 (Vitvi04g01613.t01) as a reference gene."

Q6:

Figure legend 6 “Expression of the date VvSWEET”the term “date” refers to the berry, that is the fruit unit composing the grape? Then it should be corrected.

A6:

We sincerely apologize for the misunderstanding caused by inaccurate expression. We have corrected the figure legend 6，“Expression of the VvSWEET gene family in grape fruits during the green fruit stage, veraison, and ripening based on transcriptome data.”

Q7:

In the Discussion section ln 428-433 “wheat has the highest number of SWEET members” should be elaborated to indicate that cultivated wheat is either allotetraploid (AABB) for durum wheat (Triticum turgidum ssp. durum), or allohexaploid (AABBDD) for bread wheat (Triticum aestivum), thus justifying the high number of SWEET genes identified in a species with multiple genomes.

A7:

Thank you to the reviewer for your meticulous guidance on this issue. Following your suggestion, the corresponding supplementary text has been updated and expanded in the discussion section, as detailed in “Furthermore, we discovered... of SWEET genes.”

Q8：

In ln 430 “loquat” should be followed by the species name.

A8:

Thank you to the reviewer for your attention to detail. Following your suggestion, we have added the species name “Eriobotrya japonica” after "loquat".

Q9:

Given the scrupulous bioinformatics analyses conducted in this study, I would suggest presenting a Table to depict the species assessed/compared and the number of SWEET family genes identified in each species, including the grapevine, with the relevant references.

A9:

Thank you for your suggestion. Following your advice, we have created a table (Table 1. SWEET families of several plant species) that details the number of SWEET family genes in each species, along with the relevant references for each data point.

Reviewer 3 Report

Dear authors I have reviewed your manuscript, in general is well written, some editions are required.

References 18, 19 and 25 doesnt match. Some scientific names have to be italized.

Comments for author File: Comments.pdf

Author Response

Dear editor and reviewers,

Many thanks for spending time to review our work.

We are grateful to the Editor and anonymous reviewers for their valuable comments and suggestions which help to improve the quality of the paper. We have studied the reviewers’ comments carefully and have made modifications and corrections which we hope meet their approval.

Q:

References 18, 19 and 25 doesnt match. Some scientific names have to be italized.

A:

Thank you for your suggestion. We have carefully reviewed and corrected the issues regarding the matching of references and the formatting of scientific names.