Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Excessive zinc increases reactive oxygen species production within plant cells, inhibits photosynthesis, and impairs nutrient uptake. In this manuscript, the authors employed metabolomics and proteomics to reveal the metabolic responses of young leaves, mature leaves, and roots of Amaranthus caudatus to zinc stress, providing foundational data for optimizing amaranth's nutritional growth. However, the analysis of proteomics results fails to integrate physiological and biochemical parameters to elucidate the metabolic responses of young leaves, mature leaves, and roots under zinc stress. Much of the data remains confined to pure statistical analysis, failing to reveal underlying mechanisms. Furthermore, the proteomics analysis lacks sufficient rigor. Proteomics studies should prioritize identifying differentially regulated metabolic pathways rather than focusing on individual proteins.

1. Introduction

Regarding research progress, the authors could introduce the response mechanisms of the model plant Arabidopsis thaliana (both Amaranthus caudatus and Arabidopsis belong to the dicotyledonous class Magnoliopsida) to zinc excess, providing a reference for this manuscript's research.

The authors mention that the genus Amaranthus is a zinc-accumulating plant and can serve as a phytoremediation plant for zinc-contaminated soils. Can the zinc accumulation levels in Amaranthus meet the criteria for use as a soil remediation plant for zinc-contaminated soils? While accumulating excessive zinc, does it also accumulate toxic heavy metals like cadmium? Could this pose health risks if consumed by humans?

2. Results

Recommend adding phenotyping figures for different treatments within the main text.

Basic physiological parameters under different treatments, such as photosynthetic indices, should be presented in the main text.

2.2. Protein isolation and tryptic digestion: These results represent basic proteomics data and should be simplified or presented in supplementary materials.

The proteomics analysis lacks professional rigor. Such analyses should focus on metabolic pathways involving differentially expressed proteins rather than isolated high-abundance proteins.

Proteomics results cannot merely list altered proteins; they must be interpreted in conjunction with physiological and biochemical parameters.

 

The Discussion section is extensive; streamlining is recommended.

4. Materials and Methods

4.1. Reagents: Given space constraints, omit descriptions of routine chemical and biological reagents.

4.2. Plant growth conditions and Zn stress application: This experimental methodology is critical to the results. Add details on plant growth conditions and zinc stress treatment protocols. Add descriptions of experimental treatments and sampling conditions for proteomics and metabolomics analyses.

What plant varieties were used in the author's experiments, and are they commercially available?

Is Thermo Fisher Scientific a U.S. or German company? Clarify the criteria for identifying proteins with high reliability.

Throughout the manuscript, the terminology for protein changes is inconsistent. In proteomic analysis, protein abundance changes are described as increased or decreased. Not “up-regulated” and “down-regulated.” In my opinion, “up-regulated” and “down-regulated” are terms used for gene expression. The term “differentially expressed proteins (DEPs)” is also not proper. “Differentially accumulated proteins (DAP)” is more suitable for describing differential proteins (Zheng et al. Journal of Proteomics, 2023, 280: 104894).

Numerous methods exist for metabolomics research, including GC-MS and LC-MS approaches, and method selection impacts experimental outcomes. The authors' description of their metabolomics experimental methodology is missing.

Author Response

We are thankful to the Reviewer 1 for very detailed analysis of our work and careful attention to the important details. Taking into account this valuable information, we provide our response and corresponding corrections in manuscript text on each remark.

Comment 1: Excessive zinc increases reactive oxygen species production within plant cells, inhibits photosynthesis, and impairs nutrient uptake. In this manuscript, the authors employed metabolomics and proteomics to reveal the metabolic responses of young leaves, mature leaves, and roots of Amaranthus caudatus to zinc stress, providing foundational data for optimizing amaranth's nutritional growth.

Answer: In response to this comment, we would like to clarify that in this manuscript we employed only proteomics but not metabolomics to reveal the metabolic responses of young leaves, mature leaves, and roots of Amaranthus caudatus to zinc stress. A study describing employed metabolomics, namely GC-MS-based profiling of primary metabolites of Amaranthus caudatus leaves and roots to zinc stress was published by us earlier:

Osmolovskaya, N., Bilova, T., Gurina, A., Orlova, A., Vu, V. D., Sukhikh, S., Zhilkina, T., Frolova, N., Tarakhovskaya, E., Kamionskaya, A., & Frolov, A. (2025). Metabolic Responses of Amaranthus caudatus Roots and Leaves to Zinc Stress. Plants, 14(14), 2119. https://doi.org/10.3390/plants14142119.

In the text of our present manuscript, we have often quoted this previous article. Importantly, our study had two main goals  - in addition to the first major goal of this work as " identification of the Zn-responsive proteins in the roots, young and mature leaves of the A. caudatus plants exposed to Zn stress for seven days"(line 110), the second main objective of this study was to investigate the involvement of the Zn-responsive proteins in "Zn²⁺-induced adaptive metabolic shifts discovered in these organs earlier"(lines 111-113) and described in our previous article mentioned above.

Remark 1: However, the analysis of proteomics results fails to integrate physiological and biochemical parameters to elucidate the metabolic responses of young leaves, mature leaves, and roots under zinc stress

Answer: We cannot agree with the Reviewer’s opinion. The primary focus of our study was on identifying Zn-response proteins in roots and leaves of A. caudatus plants treated with Zn, and examining whether the Zn-responsive proteins may be involved in the Zn²⁺-induced adaptive metabolic shifts that were found in these organs in our previous metabolomics article. Thus, the discussion aimed to integrate the Zn-responsive proteins and Zn²⁺-induced adaptive metabolic shifts and physiological processes that can be affected by the metabolic shifts is provided in the last subsection 3.6 (lines 837-927).

3.6. Consideration of the Zn-induced alterations in A. caudatus proteome in the context of the accompanying metabolic adjustments gives access to the mechanisms of Zn stress tolerance

and is summarized in the scheme presented in Figure 6.

In accordance with the Reviewer remark and to make the integration of proteomics results with physiological parameters clearer, we added a few text fragments (marked in bold) to highlight the potential involvement of Zn-responsive proteins in the Zn-induced changes such as visible chlorosis of young leaves:

 “The young leaves of the Zn-exposed plants which showed visible chlorosis symptoms displayed another pattern of DAPs implicated in photosynthetic ETC.” (lines 696-697)

“In general, these findings indicate that exposure to high Zn²⁺ concentrations damages photosynthetic ETC or even prevent its establishment and chlorophyll biosynthesis in the young leaves causing their chlorosis.” (lines 718-719)

 

Remark 2: Much of the data remains confined to pure statistical analysis, failing to reveal underlying mechanisms. Furthermore, the proteomics analysis lacks sufficient rigor.

Answer: We can agree with the Reviewer that “Much of the data remains confined to pure statistical analysis” if his statement refers to the Results section which involves only a description of the results but not a discussion of them. The results of proteomics analysis are further comprehensively discussed in six subsections of the Discussion, each devoted to an assigned specific protein function to reveal Zn-induced mechanisms underlying at the plant cell and organism levels.

To reach sufficient rigor in our proteomics analysis in our bioinformatics pipeline, we employed Mercator MapMan online tool, as described in Materials and methods and Results sections (lines 1006; line 378 and further). This plant-specific database maps proteins to a pre-defined list of functions, and the output is then extensively inspected and corrected manually based on literature search (see Supplementary information 5 to assess all function-specific classification). Overall result is presented in Figure 3.

Remark 3: Proteomics studies should prioritize identifying differentially regulated metabolic pathways rather than focusing on individual proteins.

Answer: In description and discussion of proteomics analysis results the main focus was paid on the characterization of individual differentially regulated proteins and their potential contribution in corresponding metabolic pathways rather than on identifying differentially regulated metabolic pathways. The reason is that this experiment revealed a relatively small number of Zn-responsive proteins (78, 40 and 21 for roots, young and mature leaves, respectively). Therefore, pathway enrichment analysis is unlikely to yield results for small sets of differentially regulated proteins with fewer than 100 in each plant organ. In accordance with the remark the following text was added to the Result section of the manuscript (lines 458-464):

Since this experiment revealed a relatively small number of Zn-responsive proteins (78, 40 and 21 for roots, young and mature leaves, respectively), pathway enrichment analysis is unlikely to yield results for such small sets of differentially regulated proteins with fewer than 100 in each plant organ. Thus, further elucidation of important Zn-regulated metabolic pathways was based on numbers of Zn-responsive proteins assigned to a specific function. This highlighted at least six major protein functions: stress response and redox metabolism, protein biosynthesis and homeostasis, photosynthesis, sugar metabolism, energy metabolism and ion transport.

Remark 4: Regarding research progress, the authors could introduce the response mechanisms of the model plant Arabidopsis thaliana (both Amaranthus caudatus and Arabidopsis belong to the dicotyledonous class Magnoliopsida) to zinc excess, providing a reference for this manuscript's research.

Answer: We agree with the Reviewer that both Amaranthus caudatus L and model plant Arabidopsis thaliana belong to the same clade Magnoliopsida. However, they belong to different orders (Amaranthus caudatus L. to Caryophyllales and Arabidopsis thaliana to Brassicales). Besides, Amaranthus plants are characterized by C4 photosynthetic pathway, which may have an impact on their response to HM stress. According to (Remans et al., 2012) exposure of Arabidopsis thaliana to excess Zn revealed a Zn-specific oxidative stress signature that support the existence of Zn-specific signal transduction pathways influencing anti-oxidative responses. In addition, in study Fukao et al (2011) adaptive physiological responses and Zn-induced toxicity symptoms on Zn-treated Arabidopsis seedling were described and their appearance were explained by obtained proteomics results. Thus, in accordance with the remark, we have supplemented the following text (bold marked) into the Introduction section (lines 95-105):

In this context, proteomics represents a versatile tool for disclosing the molecular mechanisms behind the Zn toxicity and plant tolerance to Zn stress, as was shown by Fukao et al. (2011) in Arabidopsis thaliana seedlings, Šimon et al. (2021) in Oryza sativa roots and by Lucini et al. (2015) in Lactuca sativa leaves using gel-based and gel-free bottom-up approaches [28,29,30]. A study on A. thaliana seedlings grown under Zn excess can serve as a model to illustrate the significance of proteomics data in understanding the development of adaptive physiological reactions or Zn toxicity symptoms [Fukao et al., 2011]. The seedling toxicity symptom such as chlorosis was associated with an increase in abundance of a few proteins known to be responsive to iron deficiency, indicating that Zn displaces iron from active sites of enzymes involved in chlorophyll synthesis. Another A. thaliana Zn toxicity symptom is a reduction in seedling root growth was associated with decrease in abundance of a few subunits of V-ATPase which are responsible for cell expansion and Zn²⁺ ion sequestration into vacuole [Fukao et al., 2011].

References:

Remans T, Opdenakker K, Guisez Y, Carleer R, Schat H, Vangronsveld J, Cuypers A. Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature. Environmental and Experimental Botany. 2012, 84: 61–71. https://doi.org/10.1016/j.envexpbot.2012.05.005.

 

Remark 5: The authors mention that the genus Amaranthus is a zinc-accumulating plant and can serve as a phytoremediation plant for zinc-contaminated soils. Can the zinc accumulation levels in Amaranthus meet the criteria for use as a soil remediation plant for zinc-contaminated soils? While accumulating excessive zinc, does it also accumulate toxic heavy metals like cadmium? Could this pose health risks if consumed by humans?

Answer: In the Introduction we only mentioned that “The genus Amaranthus is not only a valuable source of nutrients and biologically active metabolites [Rastogi et al., 2013)], but also is a highly adaptive crop [Riggins et al., 2021] and a versatile tool for phytoremediation of Zn-contaminated agricultural soils [Lukatkin et al., 2021]”.

Regarding whether Amaranthus meets the criteria of a phytoremediation, it should be clarified that Amaranthus species attract a special interest as potential candidates for Zn phytoextraction (implies Zn accumulation in shoots) (Lukatkin et al., 2021) and Zn phytostabilization (implies immobilization in roots) (Hunkova et al., 2024). In the latter study, it was noted that Zn translocation from roots to shoots (TF =0.2-0.4) is not efficient enough to consider grain amaranth cultivars (Amaranthus cruentus and Amaranthus hypochondriacus ×Amaranthus hybridus) as a Zn hyperaccumulator. However, a strong phytostabilization potential is presumed for each examined cultivar. Furthermore, when exposed to 150 mg/L of Zn in Hoagland nutrient solution accumulation of Zn in roots of various Amaranthus cultivars reached 4446-6150 mg· kg^-1 DW and in shoots -  962–1985 mg· kg^-1 DW (Hunkova et al., 2024). The levels in shoots were much higher than maximum permissible limit 99.4 mg/kg DW set for Zn in vegetables by the Food and Agriculture Organization (FAO)/World Health Organization (WHO) to prevent toxicity and ensure food safety (Ben Chabchoubi et al., 2020, https://doi.org/10.1007/s41207-020-00193-9). Other authors (Yap et al., 2022,  https://doi.org/10.3390/biology11030389) claim that non-hyperaccumulating green amaranth Amaranthus viridis is very promising phytoextraction agent of Zn and Ni and a very promising phytostabiliser of Cd and Fe and can be used in the phytoremediation of soils polluted by Cd, Fe, Ni, and Zn  whereas (Fouad et al., 2023) concluded that Amaranthus viridis on contaminated soils is more effective both for Cd and Ni phytostabilization than phytoextraction. We did not find any works where the phytoremediation potential was studied specifically for Amaranthus caudatus, except the research on its potential as a phytoremediating agent for lead (Abubakar, 2014). However, as follows from the data obtained in our previous study (Osmolovskaya et al., 2025), A.caudatus plants, when exposed to 300 µmol/L ZnSO₄, accumulated 3531 mg· kg^-1 DW Zn in the roots and 168 and 479 mg· kg^-1 DW in mature and young leaves, what exceeds the permissible limit of Zn for crop leaves but indicates the possibility of Amaranthus caudatus using for phytostabilization purposes on Zn-polluted soils due to its high ability to immobilize Zn in the roots. It should be noted that plants used in phytoremediation are harvested and subjected to special disposal.

Since the above indicated Reviewer’s questions do not relate to the main subject of the presented study, we limited our response to the questions by only their above-mentioned discussion and have not made any relevant text supplementation in the Introduction because of space limitations of the section.

References:

Rastogi, A.; Shukla, S. Amaranth: A new millennium crop of nutraceutical values. Crit. Rev. Food Sci. Nutr. 2013, 53, 109–125. doi:10.1080/10408398.2010.517876.

Riggins, C.W.; Barba de la Rosa, A.P.; Blair, M.W.; Espitia-Rangel, E. Editorial: Amaranthus: Naturally Stress-Resistant Resources for Improved Agriculture and Human Health. Front. Plant Sci. 2021, 12, doi:10.3389/fpls.2021.726875

Lukatkin, A.S.; Bashmakov, D.I.; Al Harbawee, W.E.Q.; Teixeira da Silva, J.A. Assessment of Physiological and Biochemical Responses of Amaranthus Retroflexus Seedlings to the Accumulation of Heavy Metals with Regards to Phytoremediation Potential. Int J Phytoremediation. 2021, 23, 219–230, doi:10.1080/15226514.2020.1807904.

Hunková J., Lisinovičová M., Lancíková V., Szabóová M., Kačírová J., Mistríková V., Hricová A. A comparative analysis of heavy metal stress responses in different grain amaranth cultivars. Plant Stress, 2024, Volume 14, 100619, https://doi.org/10.1016/j.stress.2024.100619.

Ben Chabchoubi, I., Mtibaa, S., Ksibi, M. et al. Health risk assessment of heavy metals (Cu, Zn, and Mn) in wild oat grown in soils amended with sediment dredged from the Joumine Dam in Bizerte, Tunisia. Euro-Mediterr J Environ Integr. 2020, 5, 60 . https://doi.org/10.1007/s41207-020-00193-9

Yap, C.K.; Yaacob, A.; Tan, W.S.; Al-Mutairi, K.A.; Cheng, W.H.; Wong, K.W.; Berandah Edward, F.; Ismail, M.S.; You, C.-F.; Chew, W.; et al. Potentially Toxic Metals in the High-Biomass Non-Hyperaccumulating Plant Amaranthus viridis: Human Health Risks and Phytoremediation Potentials. Biology.2022, 11, 389. https://doi.org/10.3390/biology11030389

Fouad M. S., Megahed M. A., Nabil A. Abo Hamed, Hoda F. Zahran, Abdel-NasserA. Abdel-Hafeez. Assessment of phytoremediation efficacy of Amaranthus viridis L. against cadmium and nickel. Fayoum Journal of Agricultural Research and Development. 2023, 37, 1: 63 - 81. DOI: 10.21608/fjard.2023.281078.

Abubakar M. M., Anka U. S., Ahmad M. M., Getso B. U. The Potential of Amaranthus caudatus as a Phytoremediating Agent for Lead. Journal of Environment and Earth Science. 2014. Vol.4, No.10,  www.iiste.org. ISSN 2224-3216 (Paper) ISSN 2225-0948

 

Remark 6: Recommend adding phenotyping figures for different treatments within the main text. Basic physiological parameters under different treatments, such as photosynthetic indices, should be presented in the main text.

Answer: The corresponding figures demonstrating Zn-treated and control plants and their basic physiological parameters (stomatal conductivity, chlorophyll content, photosystem II (PS II) activity, leaf relative water content) and their description were added to the main text as Figure 1 (lines 131), namely, physiological plots and photos of typic plants. Therefore, numeration of other figures has been changed, references to the figures were revised.

Remark 7: Protein isolation and tryptic digestion: These results represent basic proteomics data and should be simplified or presented in supplementary materials.

Answer: According to the Reviewer recommendation the part presenting description of the protein isolation and tryptic digestion was shortened to remove facultative information (lines 151-157). The full lists of protein yields and densitometric analysis data are presented in Supplementary information 1.

 

Remark 8: The Discussion section is extensive; streamlining is recommended.

Answer: According to the Reviewer recommendation we have revised the Discussion and tried to make this section more concise to point out only the main metabolic processes affected by Zn excess exposure. All changes made in the text are highlighted with yellow.

 

Remark 9: 4.1. Reagents: Given space constraints, omit descriptions of routine chemical and biological reagents.

Answer: According to the Reviewer recommendation, we decided to move this section in Supplementary information 1 in the new section named “Materials and reagents”, page 5

 

Remark 10: 4.2. Plant growth conditions and Zn stress application: This experimental methodology is critical to the results. Add details on plant growth conditions and zinc stress treatment protocols. Add descriptions of experimental treatments and sampling conditions for proteomics and metabolomics analyses.

What plant varieties were used in the author's experiments, and are they commercially available?

Answer: Detailed information on growth conditions and stress application is now provided in section 4.1 (lines 929-961). We also added details on nutrient solution composition in Protocol S1-1, Supplementary information 1 (page 5) and renumerated other protocols (Supplementary information 1, page 6; main text line 972).

Amaranthus caudatus L. var. Karwa dauta was used in these experiments, which is not widely or commercially available. The seeds for the experiment were provided by request from seed collections of Vavilov All-Russia Institute of Plant Genetic Resources as indicated in lines 931-933 and 1062-1064.

 

Remark 11: Is Thermo Fisher Scientific a U.S. or German company?

Answer: Related parts were clarified in the text, pointing out that this company is now based in USA (lines 983, 987-989).

 

Remark 12: Clarify the criteria for identifying proteins with high reliability.

Answer: Protein identification relied on Proteome Discoverer software is described in section 4.4. Details for search settings are listed in table S1-11. We also added clarification on FDR threshold in the main text and related table (table S1-11, page 23; main text lines 998-999). To filter only high-confident identifications, internal post-processing algorithm Percolator was also employed as a semi-supervised machine learning algorithm to improve confidence of annotations (J. Proteome Res. 2009, 8, 7, 3737–3745, https://doi.org/10.1021/pr801109k).

 

Remark 13: Throughout the manuscript, the terminology for protein changes is inconsistent. In proteomic analysis, protein abundance changes are described as increased or decreased. Not “up-regulated” and “down-regulated.” In my opinion, “up-regulated” and “down-regulated” are terms used for gene expression. The term “differentially expressed proteins (DEPs)” is also not proper. “Differentially accumulated proteins (DAP)” is more suitable for describing differential proteins (Zheng et al. Journal of Proteomics, 2023, 280: 104894).

Answer: We are grateful for careful attention for this principal detail. Despite the wide utilization in academic literature these terms in relation to protein abundance levels, it would be more precise to replace our terms with your suggestions, which therefore was made (lines 233, 269, 290, 294, 296, 298, 299, 301, 303, 306, 318, 319, 325, 328, 330, 333, 339, 350, 357, 358, 360, 367,378, 402, 403, 414, 445, 454, 465, 490, 492).

On behalf of all co-authors

Corresponding author,

Dr. Tatiana Bilova

Reviewer 2 Report

Comments and Suggestions for Authors

In the present work  a Zn induced stress was analyzed by using proteomics approach.

Abstracts

Line 21-23:  “Hence, we aimed to investigate the complexity of metabolic responses to 21 Zn stress in Amaranthus caudatus young and mature leaves, as well as in roots by means 22 of metabolomics and proteomics”. The authors should be separated the metabolomics analysis obtained in previous work from proteomics analysed in the present work.

Results,

In table 1, The column FC corresponds to Fold Change, why the FC 2,2 is a reduction of the proteins indicated with a down arrow, if the author considers a FC greater than 1.5 as a protein up-accumulation.

In figure 5, which is the red boxes, it is not clear in the figure.

Methods

Line 933-935: Please describe shortly the plant growth conditions and Zn stress application, and them site the article

Line 954:” The dried tryptic hydrolysates were sequentially reconstituted in 60, 20 and 3% (v/v) aq. acetonitrile containing 0.1% (v/v) formic acid”; this finally is only one sample injected in the LC-MS/MS.

It is recommended that the authors deposite the raw data in some of the repositories included in the https://www.proteomexchange.org/

In general, the results are very interesting and will contribute to metal stress, but the authors will improve the structure of data presentation, the data presented in the table will be clarified, wich proteins are increased or which proteins are decreasing by stress.

Author Response

We thank Reviewer 2 for reading our work and valuable comments on the text of the article. Based on important notes and corrections, we improved some details in the manuscript.

 

Remark 1: Line 21-23: “Hence, we aimed to investigate the complexity of metabolic responses to 21 Zn stress in Amaranthus caudatus young and mature leaves, as well as in roots by means 22 of metabolomics and proteomics”. The authors should be separated the metabolomics analysis obtained in previous work from proteomics analysed in the present work.

Answer: Thank you for this note, we edited this passage to clarify the message: Hence, we aimed to investigate the complexity of metabolic responses to Zn stress in Amaranthus caudatus young and mature leaves, as well as in roots by means of proteomics. Our previous metabolomics research has indicated potential involvement of gluconate and salicylate in Zn tolerance mechanisms (lines 22-23).

 

Remark 2: In table 1, The column FC corresponds to Fold Change, why the FC 2,2 is a reduction of the proteins indicated with a down arrow, if the author considers a FC greater than 1.5 as a protein up-accumulation.

Answer: Fold Change (FC) stands for magnitude of abundance changes of particular protein among two treatment groups. That is, this applies to both increase (↑) and decrease (↓) of the protein levels in experimental group in comparison to control. Corresponding clarifications were added to the text.

 

Remark 3: In figure 5, which is the red boxes, it is not clear in the figure.

Answer: In the description to the Figure 5, it was indicated that “The observed here proteomic findings are indicated with green (up-regulation) and red (down-regulation) rectangles” (lines 853, 854)

 

Remark 4: Line 933-935: Please describe shortly the plant growth conditions and Zn stress application, and them site the article

Answer: We added this information in the corresponding section 4.1 Plant growth conditions and Zn stress application (line 929)

 

Remark 5: Line 954:” The dried tryptic hydrolysates were sequentially reconstituted in 60, 20 and 3% (v/v) aq. acetonitrile containing 0.1% (v/v) formic acid”; this finally is only one sample injected in the LC-MS/MS.

Answer: Text has been revised and modified (marked in bold) to clarify this aspect (lines 980-982):

Each of the sample containing dried tryptic hydrolysates were sequentially reconstituted in 60, 20 and 3% (v/v) aq. acetonitrile containing 0.1% (v/v) formic acid, and resulted samples were loaded on an Acclaim PepMap trap column…

 

On behalf of all co-authors,

Corresponding author,

Dr. Tatiana Bilova

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript was modified.

Author Response

We highly appreciate Reviewer 1 for evaluating our revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The improvements to the manuscript will allow for a better understanding of the data

However, a small correction is needed.  The FC value represents a ratio, in this case between the Zn-treated sample and the control. When the value is greater than 1, it indicates an increase; if it is lower than 1, it indicates a decrease. In this study, a fold change of 1.5 was considered a significant increase or decrease. The authors adjusted the FC values for cases showing a decrease by presenting the inverse of the FC value. This should be explained in the methodology section or in the table footnote.

Author Response

We are thankful to Reviewer 2 for evaluating our revised manuscript and for pointing out the important remark below.

Remark 1: However, a small correction is needed.  The FC value represents a ratio, in this case between the Zn-treated sample and the control. When the value is greater than 1, it indicates an increase; if it is lower than 1, it indicates a decrease. In this study, a fold change of 1.5 was considered a significant increase or decrease. The authors adjusted the FC values for cases showing a decrease by presenting the inverse of the FC value. This should be explained in the methodology section or in the table footnote.

Answer: As suggested by Reviewer we added clarifying corrections for the fold change in the section Materials and methods (lines 1008-1019):

“To select proteins that are significantly differentially abundant, thresholds for FC and FDR adjusted p-value were set at 1.5 as the lowest value and 0.05 as the highest value, respectively. With this, proteins that showed a greater than 1.5-fold increase in abundance under Zn stress compared to the control (FC(Zn-stress/Control)≥1.5), were considered as up-accumulated; and proteins that showed a greater than 1.5-fold decrease in abundance under Zn stress compared to the control (FC(Control/Zn-stress)≥1.5) were considered to be down-accumulated.”

and in the Tables 1-3 footnote (lines 314-317, 349-353, 380-384):

 “…for proteins that demonstrated an increase in abundance, FC(Zn stress/Control) indicates an (≥1.5-fold) increase in their abundance under stress Zn compared to the control, and for proteins that showed a decrease in abundance, FC(Control/Zn stress) shows a (≥1.5-fold) decrease in their abundance under Zn stress compared to the control.”

 and we also included two additional lines in the Tables 1-3 to categorize the proteins that were differentially abundant into two groups:

“Proteins displaying a higher abundance under Zn stress in comparison to control”

and

“Proteins displaying a decreased abundance under Zn stress in comparison to control”

 

We hope that these adjustments will make it easier to understand how we used the inverse values of FC to demonstrate a reduction in abundance of specific proteins during stress Zn.

On behalf of all co-authors,

Dr. Tatiana Bilova