Comparative Omics-Based Identification and Expression Analysis of a Two-Component System in Vigna radiata in Drought Stress

Round 1

Reviewer 1 Report

Line 279 ... on chromosomes 1-10 (Chr 1–10).

Line 280, preferably do not use the "aa" notation, use the full name "amino acids"

Line 282, indicate the units of molecular weight

Table 2, titles: Isoelectric point (PI), Protein length (amino acids), No. of exons

Line 431, remove parentheses of CK18)

Write the full species name at the first occurrence in the text, and then write the abbreviated species. Because they appear indistinctly written in full or abbreviated throughout the body of the text

I think you should consider including in the title the following: Comparative Omics-based identification and expression analysis of the two-component system in Vigna radiata and its response to drought

 

 

Author Response

Reviewer 1-

Comments and Suggestions for Authors

Line 279 ... on chromosomes 1-10 (Chr 1–10).

Our response: Thanks for the suggestion. We have revised the line “VrTCSs are found on chromosomes 1-10.”

Line 280, preferably do not use the "aa" notation, use the full name "amino acids"

Our response: Thanks for the suggestion. We have removed the “aa” notation of amino acids. And the revised line is “. The length of the proteins ranged from 111 amino acids (VrHP5) to 1265 amino acids (VrHk1.1)”.

Line 282, indicate the units of molecular weight

Our Response: Thanks for the suggestion. We have now introduced the unit of molecular weight of a protein.

Table 2, titles: Isoelectric point (PI), Protein length (amino acids), No. of exons

Our Response: Thanks for the recommendation. Now, we have corrected the titles of the table 2.

Line 431, remove parentheses of CK18)

Our Response: Thanks for the recommendation. Now, we have removed the parentheses of CK18.

Write the full species name at the first occurrence in the text, and then write the abbreviated species. Because they appear indistinctly written in full or abbreviated throughout the body of the text

Our Response: Thanks for the recommendation. We have carefully checked the abbreviation of species name and as per recommendation we have corrected all the abbreviation throughout the text.

I think you should consider including in the title the following: Comparative Omics-based identification and expression analysis of the two-component system in Vigna radiata and its response to drought

Our Response: Dear reviewer, we have updated the title as advised. Now the title appears as follows.

Comparative Omics-based identification and expression analysis of the two-component system in Vigna radiata in drought

Reviewer 2 Report

This study identified the TCS in V. radiata together with expression studies. The authors show that TCS genes in V. radiata are randomly dispersed in the chromosomes and that its products have closer evolutionary relationships with other legume genes. They also classified the proteins according to their structural domains and predicted putative regulatory motifs in the promoter regions. Finally, they analyzed the TCS expression patterns in response to drought conditions. The results of this work are important for the area and the paper is interesting and reasonably well-written.

 

 

The authors should be careful that not all readers know the similarities and differences between the TCS in prokaryotes and eukaryotes. Therefore, the authors can consider including in the introduction that in bacterium the most common TCS is the simpler version HK-RR, but that also exist the multistep phosphorelay that uses multistep His-Asp-His-Asp relay mechanism where an HK-HPt-RR participates.

 

Regarding drought stress, authors refer as inadequate water and well-watered supply. However, drought conditions are defined by leaf water potential, and the authors do not mention this method.  It is also unclear when the drought regime began applied: from the beginning or a specific time after cultivation?

 

Some abbreviations used are not specified in the text. For example, in line 194, VrHKLs means that HKs are HK-like? In the same sense, the authors define PPR up to line 356 but the abbreviation is used much earlier in the text.

 

In line 204 it should be phosphoreceptor instead of photoreceptor.

 

Some figures are difficult to read. For example, in figure 3, the evolutionary analysis could be represented by component instead of all three components in the same figure.

 

In the discussion, the authors mention: “VrHK2, VrHK3, VrHK1.2, and ethylene receptors were found to be upregulated in seeds” (line 555). However, in materials and methods, authors described that they used 28 days old plants and never mentioned something about seeds.

 

 

Author Response

Reviewer 2

Comments and Suggestions for Authors

This study identified the TCS in V. radiata together with expression studies. The authors show that TCS genes in V. radiata are randomly dispersed in the chromosomes and that its products have closer evolutionary relationships with other legume genes. They also classified the proteins according to their structural domains and predicted putative regulatory motifs in the promoter regions. Finally, they analyzed the TCS expression patterns in response to drought conditions. The results of this work are important for the area and the paper is interesting and reasonably well-written.

 

Our response: Thank you very much for your positive comment.

 

 

The authors should be careful that not all readers know the similarities and differences between the TCS in prokaryotes and eukaryotes. Therefore, the authors can consider including in the introduction that in bacterium the most common TCS is the simpler version HK-RR, but that also exist the multistep phosphorelay that uses multistep His-Asp-His-Asp relay mechanism where an HK-HPt-RR participates.

 

Our response: Thank you very much for your positive comment.

 

Regarding drought stress, authors refer as inadequate water and well-watered supply. However, drought conditions are defined by leaf water potential, and the authors do not mention this method.  It is also unclear when the drought regime began applied: from the beginning or a specific time after cultivation?

 

Our Response: Thanks for the suggestion. It is now revised as” After the cultivation of 28 days the drought stress were applied on treatment pods in which complete stop of water supply  Leaf samples were gathered from each pot (Control, and Drought) at 0 day, 3^rd day and 5^th day with three biological replicates for RNA extraction.”

 

Some abbreviations used are not specified in the text. For example, in line 194, VrHKLs means that HKs are HK-like? In the same sense, the authors define PPR up to line 356 but the abbreviation is used much earlier in the text.

Our Response: Thanks for the suggestion. Now we have add the abbreavation of VrHKls as “Histidine kinase like” . In introduction line 77 discuss about the PRR as “Moreover, there is a different class of RRs, generally called pseudo-RRs (PRRs) . PRRs are not considered true RRs because they lack DDK-conserved motifs, in which essential residues for phosphorylation are absent”.

 

In n line 204 it should be phosphoreceptor instead of photoreceptor.

 . We have now improved the sentence as ”each of the nine members possessed a conserved RR (Rec) domain containing a highly conserved Asp residue that functions as a phosphoreceptor.”

Some figures are difficult to read. For example, in figure 3, the evolutionary analysis could be represented by component instead of all three components in the same figure.

Our Response: Thanks for the suggestion. In supplementary figures,  the evolutionary analysis of all the three component HK, HPT and RR were presented in separated figures.  Fig5 represent the HK evolutionary analysis, Fig 6 represent the HPt evolutionary analysis, and the Fig7 represent the evolutionary analysis of RR. Kindly check it. Thanks.

 

In the discussion, the authors mention: “VrHK2, VrHK3, VrHK1.2, and ethylene receptors were found to be upregulated in seeds” (line 555). However, in materials and methods, authors described that they used 28 days old plants and never mentioned something about seeds.

 

Our Response: Thanks for the suggestion. These results come from the  in-silico NGS RNA-seq analysis which was carried out on seed. The section 2.5 discuss about the RNA seq analysis on seed their bio project ID no. which were freely available on SRA database of NCBI.

Reviewer 3 Report

This manuscript investigates an important role of two component system genes and their role in different environmental factors, drought response being one of the main ones, in Vigna radiata (also known as Mung beans). I find the study well designed and the findings quite interesting. A good use of both mining publicly available RNA-seq datasets along with real lab-based validation of results.

However, the authors should add some specific details so that the experiments are reproducible – this mainly from the method section. Namely –

1.      Cutoff values for RNA-seq differential expression analysis – what fold change was the cutoff to categorize a gene or transcript being differentially expressed? Was P-values or Adjusted P-values included as part of the cutoff as well? If so, what value was used?

2.      Generally, the different parameters or settings for all software or bioinformatics tool used in sections 2.1, 2.2, 2.3, 2.4 and 2.5 should be included – even if it’s the default settings.

3.      What does “drought stress” mean? How is this different in moisture or water level from the control? Some metrics to measure the amount of water or moisture in both sample sets should be included.

Author Response

Reviewer 3

Comments and Suggestions for Authors

This manuscript investigates an important role of two component system genes and their role in different environmental factors, drought response being one of the main ones, in Vigna radiata (also known as Mung beans). I find the study well designed and the findings quite interesting. A good use of both mining publicly available RNA-seq datasets along with real lab-based validation of results.

            Our response: Thank you very much for your positive comment

However, the authors should add some specific details so that the experiments are reproducible – this mainly from the method section. Namely –

1. Cutoff values for RNA-seq differential expression analysis – what fold change was the cutoff to categorize a gene or transcript being differentially expressed? Was P-values or Adjusted P-values included as part of the cutoff as well? If so, what value was used?

Our Response: Dear reviewer, the statistical analyses and measures for RNA seq data quality control and differential analysis were the same as defined by the source study i.e., BioProject: PRJNA327304. For the sake of the readers, we have now mentioned this in the methods section.

2. Generally, the different parameters or settings for all software or bioinformatics tool used in sections 2.1, 2.2, 2.3, 2.4 and 2.5 should be included – even if it’s the default settings.

            Our Response: Thanks for the suggestion. Now, we have added the missing parameter of the tool that utilized.in line 109 “The parameters of BLASTp were set as non-redundant protein sequence database, and BLOSUM62 set as scoring parameter”.  In line 122“The parameter of CELLO V.2.5 were set as protein fasta sequences and choose organism as eukaryotes.” In line “The prediction range of the motif was fixed to twenty and the other thresholds were kept at their default values as classic mode for motif discovery, and minimum width of motif should be 6 and 60 respectively.”

3. What does “drought stress” mean? How is this different in moisture or water level from the control? Some metrics to measure the amount of water or moisture in both sample sets should be included.

Our Response: Dear reviewer, the drought stress treatment has been updated in the methods section. Now it appears as below.

“Plants of V. radiata were cultivated in a growth chamber for 28 days under the following conditions. The temperature ranged from 25 ℃ – 27 ℃ throughout the experimental duration, whereas the light/dark cycle was for 16/8 h. The relative humidity level was maintained at 65%. Twenty-eight days after the cultivation, the drought stress was applied. For the drought treatment, the watering to the selected pots was stopped. The pots in control were watered to normal field capacity i.e., 80-100%. Leaf samples were collected from each pot (control, and drought at 0 day, 3^rd day and 5^th day with three biological replicates for RNA extraction.”