Genome-Wide Identification of Strawberry Metal Tolerance Proteins and Their Expression under Cadmium Toxicity
, Songtao Jiu 3, Muqaddas Aleem 4, Waqar Shafqat 5, Komal Shoukat 6, Nadeem Khan 7, Muhammad Jafar Jaskani 8, Summar A. Naqvi 8,*, Sezai Ercisli 9, Amine Assouguem 10,*, Mohammed Kara 11, Riaz Ullah 12, Maha Aljabri 13 and Sameer H. Qari 14Round 1
Reviewer 1 Report
This manuscript presents the study of metal tolerance proteins (FvMTPs) and their expressional profiling under cadmium toxicity in Fragaria vesca. The topic of the manuscript fits within the scope of the Horticulturae journal and it is an interesting contribution to scientific knowledge in the field of plant physiology and plant molecular biology, in particular, of studies of plant resistance to heavy metal stress.
The manuscript needs however some revisions. Please reexamine the manuscript taking into account the following:
1) Abstract: Please add to the abstract the scientific Latin name of strawberry by the first mention. It is necessary for easier reading of genes’ names.
2) L168-177: Please specify whether plants in the experiment were grown in soil or as soilless culture (in the nutrient solution)? The concentration of Cd equal to 0.1 M is quite a high even when compared to the concentration of macronutrients (e.g. nitrogen) that are part of the nutrient solution. Such a concentration could cause not only stress associated with cadmium toxicity, but also salt stress in plants.
3) L208: A. thaliana should be italic. Please check throughout manuscript.
4) L273-274: Figure 2 legend. I suggest changing the legend to “Motif structure (A) and their gene structure analysis (B) in strawberry”. In present version, it is not clear what is indicate as “A” and as “B”.
5) L 282-294 and then until the end of the Section: Please make not italic.
6) L 288: Please check the phrase “grapevine leaves”. Is it the data from previously paper? If “yes” please add reference.
7) The quality of Figure 5 is unsatisfactory. The names of genes and values are not readable. Please revised the Figure.
8) Figure 6: Please indicate in legend what means the letters “a”, “b” etc on Figure 6A. Why there is no letters indicating the significance of difference on Figure 6B? What is the units for the “DAB staining quantifications”?
Author Response
Dear Reviewer,
We are happy that our manuscript will be reconsider for publication in your Journal.
We are thanks to Reviewer #1 and Reviewer #2 for their valuable comments and corrections.
We have carefully reviewed each of the points raised in the evaluation process,
Our corrections to Reviewer #1 and Reviewer #2 are shown yellow and light blue highlight on revised paper
Further details are given below. If you have any question, please contact me by email.
Reviewer#1
This manuscript presents the study of metal tolerance proteins (FvMTPs) and their expressional profiling under cadmium toxicity in Fragaria vesca. The topic of the manuscript fits within the scope of the Horticulturae journal and it is an interesting contribution to scientific knowledge in the field of plant physiology and plant molecular biology, in particular, of studies of plant resistance to heavy metal stress.
The manuscript needs however some revisions. Please reexamine the manuscript taking into account the following:
1) Abstract: Please add to the abstract the scientific Latin name of strawberry by the first mention. It is necessary for easier reading of genes’ names.
Response: Thank you. We have added the scientific name of strawberry (Fragaria x annanassa Duch.) in the abstract (Line 40).
2) L168-177: Please specify whether plants in the experiment were grown in soil or as soilless culture (in the nutrient solution)? The concentration of Cd equal to 0.1 M is quite a high even when compared to the concentration of macronutrients (e.g. nitrogen) that are part of the nutrient solution. Such a concentration could cause not only stress associated with cadmium toxicity, but also salt stress in plants.
Response: Thank you for this important query. The strawberry plants were kept in a medium of soil:peat:sand at 3:1:1 volume (Line 170)
Regarding Cd concentration, we made a typo error. Here we used 0.1 mM (Line 174) concentration calibrated from the already published paper by (Nosek et al., 2020).
Nosek M, Kaczmarczyk A, Jędrzejczyk RJ, Supel P, Kaszycki P, Miszalski Z. Expression of Genes Involved in Heavy Metal Trafficking in Plants Exposed to Salinity Stress and Elevated Cd Concentrations. Plants. 2020; 9(4):475. https://doi.org/10.3390/plants9040475
3) L208: A. thaliana should be italic. Please check throughout manuscript.
Response: Thank you for highlighting this issue. We have carefully checked and revised all the problems related to the italic scientific and gene names.
4) L273-274: Figure 2 legend. I suggest changing the legend to “Motif structure (A) and their gene structure analysis (B) in strawberry”. In present version, it is not clear what is indicate as “A” and as “B”.
Response: We have revised accordingly as per your suggestion.
5) L 282-294 and then until the end of the Section: Please make not italic.
Response: Thank you. We have changed the respective paragraph in non-italic text (Line 284-296).
6) L 288: Please check the phrase “grapevine leaves”. Is it the data from previously paper? If “yes” please add reference.
Response: Thank you for bringing this problem out. This is typo error and we have replaced the word from grapevine to strawberry (Line 291).
7) The quality of Figure 5 is unsatisfactory. The names of genes and values are not readable. Please revised the Figure.
Response: Thank you for this important suggestion. We have replaced the figure 5 with modified and enhanced quality figure. Hopefully, this figure will show better display in PDF version of the manuscript.
8) Figure 6: Please indicate in legend what means the letters “a”, “b” etc on Figure 6A. Why there is no letters indicating the significance of difference on Figure 6B? What is the units for the “DAB staining quantifications”?
Response: Line (313) We have modified the figure legends as per your suggestion.
In Figure 6A, we performed DAB staining which indicates brown spots accumulation with respect to alleviated duration. In Figure 6B, we counted those brown precipitates using ImageJ program. We apologize for our mistake, I have analyzed the data for the significance analysis. New figure is attached with significance level above bars. As DAB quantification is number based quantification, I didn’t find any unit of it.
Sincerely
Reviewer 2 Report
　This manuscript describes “Genome-wide identification of strawberry metal tolerance proteins (FvMTPs) and their expressional profiling under cadmium toxicity.” Although this manuscript provides several interesting scientific results, it should be prepared more adequately before acceptance for publication in the Journal “horticulturae.” There are some suggestions which would improve the quality of this manuscript in an attachment file.
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer,
We are happy that our manuscript will be reconsider for publication in your Journal.
We are thanks to Reviewer #2 for their valuable comments and corrections.
We have carefully reviewed each of the points raised in the evaluation process,
Further details are given below. If you have any question, please contact me by email.
(1) L60-64; Cobalt is introduced as one of the essential elements of plants. As far as I know, cobalt is one of the useful elements in plants. Please confirm this matter.
Response: Thank you for this important highlight. We totally agree with your statement that Cobalt perform vital functions including stem growth, leaf disc expansion, etc. Our line (Line61-64) in agreement with your statement.
(2) L137-140; References are cited differently from other references. Additionally, this reference was not found in References. Please verify that references are properly cited in this manuscript.
Response: Thank you. We have replaced the reference with the most appropriate one.
(3) L203-216; The left indent is misaligned in this paragraph.
Response: Thank you. We have aligned the text uniformly.
(4) L231-233; What do motifs 1, 2, and 3 represent? Please explain them in more detail here or in "Materials and Methods" section to help the reader's understanding.
Response: Really sorry, I didn’t really get your question. Motif of protein sequences represents different biological functions. We have already added complete information regarding motif search and visualization. If there is any specific point missing in our methodology specify it, please. We would like to add more information about it.
(5) L268-271; Table S4 in this manuscript presents the list of primers used in the qPCR analysis. Please cite the appropriate experimental results.
Response: Sorry for this mistake. We have arranged the correct sequence of supplementary information in the text and in supplementary tables.
(6) L273-274; Figure 2 is inadequately explained. Please describe in more detail how the database analysis is performed and what the results of the database analysis indicated?
Response: Thank you for this query. We have reanalyzed gene structure analysis and incorporated necessary details regarding cation_efflux and cation diffusion facilitator (CDF) domains, ZT_dimer domain, and CzcD and FieF domains (Line 241-248).
(7) L279-280; What is the rationale for the selection of approximately 1600 base pair promoter sites? Please provide this rationale with proper citation of references here or in “Materials and Methods” section.
Response: thank you for pointing out this issue. Generally, 1600bp is considered as an empirical value, believed to have promoter function in this range. However, 1500-2000 bp range is considered as a general length for the promoter analysis. Our work on similar methodology has already been published:
Cui M, Haider MS, Chai P, Guo J, Du P, Li H, Dong W, Huang B, Zheng Z, Shi L, Zhang X and Han S (2021) Genome-Wide Identification and Expression Analysis of AP2/ERF Transcription Factor Related to Drought Stress in Cultivated Peanut (Arachis hypogaea L.). Front. Genet. 12:750761. doi: 10.3389/fgene.2021.750761
(8) L282-294; The fonts are all in italics in this paragraph.
Response: Thank you for highlighting this issue. We have replaced it with plain (non-italic) text. Additionally, we have revised thoroughly and removed similar type of mistakes.
(9) Figure 5; Many graphs are indicated in this Figure. It is unclear what experimental results are shown in each graph. Please show them appropriately and explain them in more detail in Figure legend.
Response: Thank you and we are in agreement with your statement. We have now attached two independent figures of which Figure 5A indicates gene expression analysis of Cd-treated strawberry leaf tissues and Figure 5B indicates gene expression analysis of Cd-treated root tissues. Hope new figure is more clear and self-explanatory.
(10) Figure 6; Please explain in more detail in Figure legend what each DAB staining result indicates.
Response: Thank you for the suggestion. We have incorporated necessary details regarding DAB staining and quantification (Line 320)
(11) Figure 6; Please explain content of graph in Figure 6B in more detail in Figure legend.
Response: Thank you. We have incorporated the required details of Figure 6B in text (Line 313-314) and in the figure legends (Line 323).
(12) Supplementary Materials; The actual Table S1-S4 differ from what are described here. Please correct them.
Response: Thank you for this important highlight. We have now arranged the supplementary tables order in manuscript and in file.
Sincerely
