Genome-Wide Analysis of NPH3/RPT2-like (NRL) Genes in Grape (Vitis vinifera L.): Their Identification, Characterization, and Different Responses to Light Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study examines the  grape's (Vitis vinifera L.)NPH3/RPT2-Like (NRL) gene family. The NRL proteins play a crucial role in the plant's response to light. Researchers identified 24 VvNRL genes in the grape genome and analysed their structure, evolutionary relationships, and expression patterns in various tissues and under different light conditions. The study revealed that genes encoding NRL proteins are unevenly distributed across 11 chromosomes, and based on phylogenetic analysis, can be categorized into six groups. The promoter regions of all 24 genes were investigated, and cis-regulatory elements within their sequences were identified. The analysis highlighted the presence of light-responsive domains. The authors also performed molecular docking predictions, which suggested interactions between VvNRL4, VvNRL6, and the LOV domains in VvPHO1 and VvPHO2. The findings, particularly the light-induced expression of VvNRL6 and the interactions between VvNRL4, VvNRL6, and phototropins, offer a solid foundation for future studies and contribute meaningfully to the fields of plant genetics and light response mechanisms.

The article encompasses a wide range of research and a substantial amount of analysed data. It is clearly and consistently written; however, some minor improvements should be made.

In subsections 3.5 to 3.8 and the first paragraphs of the discussion section, the initial paragraphs should be removed.

The sentence “the electrophoretic mobility of the NPH3 protein significantly increases after blue light stimulation” (subsection 3.6, lines 346-347) should be elaborated on for clarity.

The sentence “Additionally, genes within the VvNRLs protein-protein interaction (PPI) network are enriched in the blue light response pathway” (page 14, lines 416-418; subsection 3.7) should be rewritten for better clarity.

The sentence “The NRL gene family is functionally associated with AGCVIII kinases and participates in gravity response by redundantly regulating organ development and auxin transport” (page 19, lines 565-567; section: Discussion) requires clarification.

On page 8 line 253 (subsection 3.2) and page 14 line 415 (subsection 3.7), it should mention AtNPH3 instead of AtNP3.

Specific conventions concerning protein and gene abbreviations should be followed to ensure clarity and consistency. For plant species, protein abbreviations are typically written in all uppercase letters and are not italicized. Gene symbols, on the other hand, are usually italicized and written in uppercase letters or with the first letter uppercase and the rest lowercase. This distinction enhances comprehension of presented data and findings, improving the overall understanding of the article.

After implementing these improvements, I recommend the article for publication in Horticulturae.

Author Response

Dear editor:

We greatly appreciate the efforts of the editor and all reviewers for the constructive comments that have helped shape this manuscript into a better form. We have addressed all the concerns by either editing the manuscript or re-drawing the figures. Please see the detailed point-by-point response added below. Changes in the manuscript have been marked in red.Formatting changes such as italics and spaces are not fully marked.

Sincerely,

Hongxia Zhang and Nianwei Qiu

 

Point-by-point response to Reviewer #1:

General comments:

This study examines the grape's (Vitis vinifera L.) NPH3/RPT2-Like (NRL) gene family. The NRL proteins play a crucial role in the plant's response to light. ..... The findings, particularly the light-induced expression of VvNRL6 and the interactions between VvNRL4, VvNRL6, and phototropins, offer a solid foundation for future studies and contribute meaningfully to the fields of plant genetics and light response mechanisms. The article encompasses a wide range of research and a substantial amount of analysed data. It is clearly and consistently written; however, some minor improvements should be made.

Response: Thank you for your comments.

 

Specific comments:

• In subsections 3.5 to 3.8 and the first paragraphs of the discussion section, the initial paragraphs should be removed.

Response: Following your suggestion, we have removed subsections 3.5 to 3.8 and the initial paragraphs of the discussion section.

• The sentence “the electrophoretic mobility of the NPH3 protein significantly increases after blue light stimulation” (subsection 3.6, lines 346-347) should be elaborated on for clarity.

Response: Thanks. Following your suggestion, we have elaborated this sentence to clarify that blue light stimulation likely induced conformational changes in the NPH3 protein, leading to a shift in its electrophoretic mobility. This change could be a result of post-translational modifications, such as phosphorylation, which are known to affect protein mobility in electrophoresis.

• The sentence “Additionally, genes within the VvNRLs protein-protein interaction (PPI) network are enriched in the blue light response pathway” (page 14, lines 416-418; subsection 3.7) should be rewritten for better clarity.

Response: Thanks. We have rewritten this sentence for better explain how VvNRL genes, through interactions within the PPI network, regulate the blue light response pathway. Since this conclusion is primarily based on data from Figure 7b, we have referenced it in the revised version for better clarity.

• The sentence “The NRL gene family is functionally associated with AGCVIII kinases and participates in gravity response by redundantly regulating organ development and auxin transport” (page 19, lines 565-567; section: Discussion) requires clarification.

Response: Thanks. We have carefully reconsidered our terminology and replaced "redundantly regulate" with "cooperatively regulate" to more accurately reflect the collaborative nature of NRL gene family members in regulating organ development and auxin transport.

• On page 8 line 253 (subsection 3.2) and page 14 line 415 (subsection 3.7), it should mention AtNPH3 instead of AtNP3.

Response: Thanks. We have replaced "AtNP3" with AtNPH3.

• Specific conventions concerning protein and gene abbreviations should be followed to ensure clarity and consistency. For plant species, protein abbreviations are typically written in all uppercase letters and are not italicized. Gene symbols, on the other hand, are usually italicized and written in uppercase letters or with the first letter uppercase and the rest lowercase. This distinction enhances comprehension of presented data and findings, improving the overall understanding of the article.

Response: Thank you for your comment. We have check through the whole manuscript to make sure that specific conventions concerning protein and gene abbreviations were followed.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript concerns an important topic concerning the functioning of plants in conditions of variable exposure to different light spectrums, which is of great importance for growth and, in the case of many plants, also the transition from the vegetative to the generative phase. The manuscript requires refinement, especially the discussion. The authors conducted numerous in silico analyses enabling the characterization of the studied gene family

 

General comments

Check the gene names throughout the manuscript!

The Discussion chapter lacks a discussion of the results concerning the promoter analysis.

The Materials and methods lack information and a detailed description of the gene expression analysis, and a list of primers used for the reaction

Please add a summary taking into account the significance of the obtained results

The list of references requires refinement, e.g. lines 693, 695 - insert bold

 

Detailed comments

Abstract

line 16 - the abbreviation of the gene name appears, the Latin name of the plant should be provided first to explain the abbreviation of the gene name VvNRL.

Introduction

Lines 38, 49, 56, 412 - insert a space between the last word of the sentence and the citation. Line 86, 106, 122, 131-132, 135, 162, 166, 181, 257, 258 – insert italics in Latin names of plants

Line 86 - provide full Latin names of plants throughout the manuscript, add Latin name maize

Materials and methods

line 111 – insert a space between the first and second sentence

line 129 - insert a space after the word “tool”

line 145 – no information on the RNA extraction method and its storage after extraction

line 155 – insert italics in gene names

lines 183, 185 - insert a space before citation

paragraph 3.3. lines 262-273 – correct gene names – add italics

line 275 – start sentence with capital letter

paragraph 3.4 and 3.5 – remove periods from end of sentence – chapter titles.

Author Response

Dear editor:

We greatly appreciate the efforts of the editor and all reviewers for the constructive comments that have helped shape this manuscript into a better form. We have addressed all the concerns by either editing the manuscript or re-drawing the figures. Please see the detailed point-by-point response added below. Changes in the manuscript have been marked in red.Formatting changes such as italics and spaces are not fully marked.

Sincerely,

Hongxia Zhang and Nianwei Qiu

 

Point-by-point response to Reviewer #2:

General comments:

The manuscript concerns an important topic concerning the functioning of plants in conditions of variable exposure to different light spectrums, which is of great importance for growth and, in the case of many plants, also the transition from the vegetative to the generative phase. The manuscript requires refinement, especially the discussion. The authors conducted numerous in silico analyses enabling the characterization of the studied gene family

Response: Thank you for your comments.

Specific comments:

• Check the gene names throughout the manuscript!

Response: Thank you. Following your suggestion, we have carefully checked the gene names throughout the manuscript and make relative revision. All gene names are consistent and correctly formatted. Italics have been applied to gene names as per standard conventions, and any inconsistencies have been corrected.

• The Discussion chapter lacks a discussion of the results concerning the promoter analysis.

Response: Thank you for your comment. We have expanded the discussion section to provide a more in-depth interpretation of the results concerning the promoter analysis, including the light-responsive cis-regulatory elements identified, to emphasize the role of these regulatory regions in light-induced gene expression and their potential impact on plant light response mechanisms.

• The Materials and methods lack information and a detailed description of the gene expression analysis, and a list of primers used for the reaction

Response: Thank you. As suggested, we have added information and a detailed description of the gene expression analysis, and a list of primers used for the reaction in the revised manuscript.

• Please add a summary taking into account the significance of the obtained results.

Response: Following your suggestion, we have added a summary taking into account the significance of the obtained results.

• The list of references requires refinement, e.g. lines 693, 695 - insert bold

Response: Thank you. We have refined the list of references as suggested, including lines 693 and 695.

• line 16 - the abbreviation of the gene name appears, the Latin name of the plant should be provided first to explain the abbreviation of the gene name VvNRL.

Response: Thank you. We have revised this sentence and provided the the Latin name of the plant first to explain the abbreviation of the gene name VvNRL.

• Lines 38, 49, 56, 412 - insert a space between the last word of the sentence and the citation. Line 86, 106, 122, 131-132, 135, 162, 166, 181, 257, 258 – insert italics in Latin names of plants

   Response: Thank you. We have revised these sentences as you suggested.

• Line 86 - provide full Latin names of plants throughout the manuscript, add Latin name maize

Response: Thank you. We have revised this as you suggested.

• line 111 – insert a space between the first and second sentence

Response: We have inserted a space between the first and second sentence.

• line 129 - insert a space after the word “tool”

   Response: We have inserted a space after the word “tool”.

• line 145 – no information on the RNA extraction method and its storage after extraction

Response: We have included detailed information on the RNA extraction method, including the protocol used and the storage conditions of the RNA samples post-extraction.

• line 155 – insert italics in gene names

Response: We have inserted italics in gene names.

• lines 183, 185 - insert a space before citation

Response: We have inserted a space before citation.

• paragraph 3.3. lines 262-273 – correct gene names – add italics

Response: We have corrected the gene names and italicized them as suggested.

• line 275 – start sentence with capital letter

   Response: We have revised this sentence as suggested.

• paragraph 3.4 and 3.5 – remove periods from end of sentence – chapter titles.

   Response: We have removed the periods at the end of the chapter titles as suggested.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

In my opinion the revised manuscript can be published in its present form.

Regards

Author Response

Please see the attachment.

Author Response File: Author Response.pdf