Morpho-Physiological and Transcriptional Regulation of Root System under Saline Conditions in Nymphaea Plants

Round 1

Reviewer 1 Report

The manuscript is not well written and contains major issues that should be rectified. The following are my comments

Title

The authors have included the common name (water lily) of the plant species in the title, although it would be more suitable if it also contained its scientific name.

Abstract

i)                   In same sentence you have used both Na+ and sodium, please abbreviate and use Na+ or use word sodium consistently throughout the paper. “The roots showed relatively high concentrations of Na+ and had the ability to hyper-accumulate sodium ions under salt stress”.

Introduction

i)                   Remove the space between numerical number and percentage unit “50 % of cultivated land will be affected by increasing salinization by the middle of the 21st century”

ii)                 The reference is a misquote as well. Be very careful while citing the reference; the cited reference should be original source of the quoted information “Currently, about 1.125 billion hectares of agricultural land are affected by salt stress, and predictions suggest that 50 % of cultivated land will be affected by increasing salinization by the middle of the 21st century”.

iii)               Arabidopsis thaliana or A. thaliana? Make consistent throughout the manuscript.

Materials and methods

i)                   You have not written about statistical analysis and software used, please write.

Discussion:

i)                   Improve the discussion portion and cite the tables and figures in this portion as well.

ii)                 Most of the references are old, please cite new references.

A minor concern

i)                   A native speaker should proofread grammar and English usage.

Author Response

To reviewer 1:

Thank you for comments and suggestions.

Q1: The authors have included the common name (water lily) of the plant species in the title, although it would be more suitable if it also contained its scientific name.

A1: We revised the title as suggested. Please check line 3 and 12.

 

Q2: In same sentence you have used both Na+ and sodium, please abbreviate and use Na+ or use word sodium consistently throughout the paper. “The roots showed relatively high concentrations of Na+ and had the ability to hyper-accumulate sodium ions under salt stress”.

A2: We used word sodium consistently as suggested. Please check line 27.

 

Q3: Remove the space between numerical number and percentage unit “50 % of cultivated land will be affected by increasing salinization by the middle of the 21st century”

A3: We removed the space as suggested. Please check line 35.

 

Q4: The reference is a misquote as well. Be very careful while citing the reference; the cited reference should be original source of the quoted information “Currently, about 1.125 billion hectares of agricultural land are affected by salt stress, and predictions suggest that 50 % of cultivated land will be affected by increasing salinization by the middle of the 21st century”.

A4: We corrected the reference. Please check line 296.

 

Q5: Arabidopsis thaliana or A. thaliana? Make consistent throughout the manuscript.

A5: We used Arabidopsis thaliana throughout the manuscript. Please check line 46.

 

Q6: You have not written about statistical analysis and software used, please write.

A6: We added statistical analysis and software. Please check line 94 and 120.

 

Q7: Improve the discussion portion and cite the tables and figures in this portion as well.

A7: We cited the tables and figures. Please check line 237, 248, 250 and 262.

 

Q8: Most of the references are old, please cite new references.

A8: As suggested, we cited several new references instead of the old ones. Please check the section of Reference, line 291-293, 300-302, 343-346, 349-351 and 357-360.

 

Q9: A native speaker should proofread grammar and English usage.

A9: The MS has been polished. The manuscript was edited for English language, grammar, punctuation, spelling and overall style by professional native-language editors at Maximum Academic Press (MAP 6601-22-12-16).

Reviewer 2 Report

I had a great opportunity to review manuscript entitled: 'Morpho-physiological and transcriptional regulation of root system under saline conditions in water lily plants' which is considered for publication in Horticulturae. The goal of this manuscript is to present and characteristics of morfo-physiological changes and transcriptome analysis in root of water lily under salt stress condition. The manuscript is elaborated on an interesting topic, but requires modifications and additions before publication.

GENERAL COMMENTS:
TITLE
The paper title is well stated, it is informative and concise.

ABSTRACT, INTRODUCTION
Abstract needs improvement (results are described too detailed). The abstract do not characterize the contents of the paper sufficiently (missing background and aims of work). The introduction was not yet well written, and it is too briefly presenting the subject and research problem.

MATERIAL AND METHODS
Material and research methods are presented appropriately and clearly. Experimental setup and the description in the methods section are well structured, and the statistical analysis is done alright. In spite of that I have a few objections against its present form. These are listed below:
2.1. Plant Materials and Salt Treatment
-This section lacks detail and is confusing. Please describe the conditions under which the plants were grown.

2.3 Transcriptome Analysis of Roots (This section lacks detail and is confusing)
-Please describe methods of RNA-seq data analysis; clean readings compared to the reference genome?  
-Please describe cluster analysis and functional annotation enrichment analysis.
-How was the expression of transcripts normalized?

RESULTS
The results obtained in this study are interesting. Results presented correctly.

Table 2. Differentially expressed genes related to the transport of ions in water lily roots under control vs. 150 mM NaCl conditions.
-Please present in the form of the list of genes showing significantly different expression in the comparison control and salt stressed plants. Separate into: down-regulated genes and up-regulated genes.
-Please present validation of RNAseq results with RT-qPCR.
-Correct 'padj' to 'p-value'.

DISCUSSION
In general, the discussion of results is correct. Please focus on the findings from the results in this research and finally answer the scientific problem.

CONCLUSIONS
This part needs to be improved. In the Conclusion I suggest to writing two other words on the aspect concerning the aspects where the future studies must be oriented.

LITERATURE
The items of literature included in the paper are rather sufficient and adequate to the subject of the paper.

The text of the manusctipt is not formatted correctly yet.
I also recommend revision of the English language by a native speaker or a commercial entity to remove minor typos in the text.

Author Response

To reviewer 2:

Q1: ABSTRACT, INTRODUCTION. Abstract needs improvement (results are described too detailed). The abstract do not characterize the contents of the paper sufficiently (missing background and aims of work). The introduction was not yet well written, and it is too briefly presenting the subject and research problem.

A1: We added background and aims of work. Please check line 13-16.

 

Q2: MATERIAL AND METHODS. 2.1. Plant Materials and Salt Treatment. This section lacks detail and is confusing. Please describe the conditions under which the plants were grown.

A2: We revised Plant Materials and Salt Treatment. Please check line 81-86.

 

Q3: 2.3 Transcriptome Analysis of Roots (This section lacks detail and is confusing). Please describe methods of RNA-seq data analysis; clean readings compared to the reference genome? 

A3: Firstly, we describe methods of RNA-seq data analysis. Please check line 103-106. Secondly, although the genome of Nymphaea colorata (Zhang et al., 2020) and Nymphaea thermarum (Povilus, et al.,2020) had been sequenced, the mapping rates of the cultivar ‘Colorado’ used in this study to the reference genome are only 10-12%. We discussed the mapping rates with professor Xu in Nanjing Agriculture University and professor Zhao in Hainan University, the mapping rates of Nymphaea lotus and Nymphaea rubra to the reference genome are both approximately 10%. In the manuscript of ‘Transcriptome Analysis Revealing the Interaction of Abscisic Acid and Cell Wall Modifications during the Flower Opening and Closing Process of Nymphaea lotus’ (Zhou et al, 2022), the transcriptome data was mapped to database Nr, Swiss-Prot, KEGG, and COG. In the under-reviewing manuscript of professor Xu in Nanjing Agriculture University, they also used Nr database as reference for water lily plants. Based on the low mapping rates to reference genome between different species, gene function was annotated based on Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO and GO database in this study.

 

Q4: Please describe cluster analysis and functional annotation enrichment analysis.

A4: We described as suggested. Please check line 103-107.

 

Q5: How was the expression of transcripts normalized?

A5: We added the method as suggested. Please check line 109-113.

 

Q6: RESULTS. Table 2. Differentially expressed genes related to the transport of ions in water lily roots under control vs. 150 mM NaCl conditions. Please present in the form of the list of genes showing significantly different expression in the comparison control and salt stressed plants. Separate into: down-regulated genes and up-regulated genes.

A6: We revised the table as suggested. Please check line 209.

 

Q7: Please present validation of RNAseq results with RT-qPCR.

A7: We presented RNAseq results. Please check line 191-193.

 

Q8:Correct 'padj' to 'p-value'.

A8: We corrected 'padj' to 'p-value' as suggested. Please check line 209.

 

Q9: CONCLUSIONS. This part needs to be improved. In the Conclusion I suggest to writing two other words on the aspect concerning the aspects where the future studies must be oriented.

A9: We improved conclusions as suggested. Please check line 272-274.

 

Q10: The text of the manusctipt is not formatted correctly yet. I also recommend revision of the English language by a native speaker or a commercial entity to remove minor typos in the text.

A10: The MS has been polished. The manuscript was edited for English language, grammar, punctuation, spelling and overall style by professional native-language editors at Maximum Academic Press (MAP 6601-22-12-16).

Round 2

Reviewer 1 Report

Manuscript Title: Morpho-physiological and transcriptional regulation of root system under saline conditions in Nymphaea plants

The authors have addressed all the comments according to suggestions. Now, it is recommended for publication after minor changes.

Line 46 and 49: You have used both Na+ and K+ directly, please abbreviate and use Na+ and K+ or use the word sodium and potassium consistently throughout the paper.

Line 63: Arabidopsis thaliana or A. thaliana? Make consistent throughout the manuscript. Still, it is not done throughout the manuscript

Author Response

Thank you for suggestions.

 

Q1:Line 46 and 49: You have used both Na+ and K+ directly, please abbreviate and use Na+ and K+ or use the word sodium and potassium consistently throughout the paper.

A1: We used Na⁺ and K⁺ consistently throughout the paper.

 

Q2:Line 63: Arabidopsis thaliana or A. thaliana? Make consistent throughout the manuscript. Still, it is not done throughout the manuscript。

A2: We used Arabidopsis thaliana throughout the manuscript. Please check line 63.

Reviewer 2 Report

This paper by Chen et al. has clearly benefited from the revision, as advised by reviewers. Moreover, I have one corrective remark:

Lines 191-193: check the correctness of figure numbering.
'To validate RNA-seq data, expression patterns of candidate genes were further analyzed using RT-qPCR (Figure 5)'. The data in figure 5 show sodium and potassium ion concentrations in water lily roots under high-salinity conditions.

Author Response

Q1:Lines 191-193: check the correctness of figure numbering. 'To validate RNA-seq data, expression patterns of candidate genes were further analyzed using RT-qPCR (Figure 5)'. The data in figure 5 show sodium and potassium ion concentrations in water lily roots under high-salinity conditions.

A1: We corrected the figure number. Please check line 193.