Physiological, Transcriptome, and Metabolome Analyses Reveal the Tolerance to Cu Toxicity in Red Macroalgae Gracilariopsis lemaneiformis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript deals with an important topic of the underlying mechanisms of copper effects on red macroalgae. Yet, the presentation of the manuscript needs wide revisions as follows:

1. The authors need to revise the manuscript according to the journal guidelines for the following errors:

- Abstract should be shortened to 200 words.

- The reference's formatting needs to be corrected as per the journal guidelines.

2. Abstract: Abstract: the background needs to be shortened. Also, there is a paucity of detail about the experimental protocol (dose of tested compounds, duration of the experiment…etc). Also, the presentation of the results in the abstract needs to be more organized.

3. Introduction:

- Lines 39-41: Heavy metals (HMs) are a prevalent source of marine pollution, noted for their high toxicity, environmental persistence, insidiousness, and bioaccumulation (Vom Endt et al., 2002; Nagajyoti et al., 2010; Zeraatkar et al., 2016). Too many references for one sentence. Cite one or two recent references. This issue has been repeated several times throughout the manuscript.

- Line 59-60: it is better to cite recent references or statistics.

4. Material and methods:

- Line 85: the justification of tested concentration selection and its relation to the real exposure scenarios need to be illustrated in more detail.

- Complete information on kits should be added as catalog number, Trademark, city, and country of origin. Also, detection range, sensitivity, inter and intra assay.

- Does data meet the assumption of homogeneity of variances and normal distribution? Clarify if the authors run a homogeneity or normality test.

5. Results:

- The exact P-value needs to be provided in the result section for a clear understanding of statistical significance.

- In all figure legends and table footnotes, the full terms of all abbreviations used should be clarified. Clarify the number of replicates n=? and means ± SD.

- Line 194-195, 197-203: should be transferred to the discussion.

Comments on the Quality of English Language

Good language.

Author Response

1. The authors need to revise the manuscript according to the journal guidelines for the following errors:

- Abstract should be shortened to 200 words.

Response: Thank you for your comments, the abstract has been shortened to 200 words.

 

- The reference's formatting needs to be corrected as per the journal guidelines.

Response: Thank you for your comments. The reference's formatting have corrected as per the journal guidelines. 

 

Abstract: Abstract: the background needs to be shortened. Also, there is a paucity of detail about the experimental protocol (dose of tested compounds, duration of the experiment…etc). Also, the presentation of the results in the abstract needs to be more organized.

Response:Thank you for your comments. The background of abstract has shortened in line 10-11 in revised manuscript and the dose of tested compounds and duration of the experiment has been added in line 13-14 in revised manuscript. The abstract has been reorganized.

 

3. Introduction:

- Lines 39-41: Heavy metals (HMs) are a prevalent source of marine pollution, noted for their high toxicity, environmental persistence, insidiousness, and bioaccumulation (Vom Endt et al., 2002; Nagajyoti et al., 2010; Zeraatkar et al., 2016). Too many references for one sentence. Cite one or two recent references. This issue has been repeated several times throughout the manuscript.

Response:Thank you for your comments. We have deleted one reference for this sentence and modified in line 294, 344 in discussion section in revised manuscript.

 

- Line 59-60: it is better to cite recent references or statistics.

Response:Thank you for your comments. We have cited recent references in line 60-61 in revised manuscript.

 

 

4. Material and methods:

- Line 85: the justification of tested concentration selection and its relation to the real exposure scenarios need to be illustrated in more detail.

Response:Thank you for your comments. The justification of tested concentration selection and its relation to the real exposure scenarios was added in line 378-388 in revised manuscript.

 

- Complete information on kits should be added as catalog number, Trademark, city, and country of origin. Also, detection range, sensitivity, inter and intra assay.

Response: Thank you for your comments. The catalog number, Trademark, city, and country of origin of kits was provided in Material and methods, as well as detection range, sensitivity, inter and intra assay of Nitrate reductase activity, and the detection limit of the Plant flavonoids test kit.

 - Does data meet the assumption of homogeneity of variances and normal distribution? Clarify if the authors run a homogeneity or normality test.

Response: Thank you for your comments. In the analysis of variance by SPSS 19.0, we chose homogeneity test and the data meet the assumption of homogeneity of variances and normal distribution.

 

5. Results:

- The exact P-value needs to be provided in the result section for a clear understanding of statistical significance.

Response: Thank you for your comments. We have provided the exact P-value in the result section.

 

- In all figure legends and table footnotes, the full terms of all abbreviations used should be clarified. Clarify the number of replicates n=? and means ± SD.

Response: Thank you for your comments. The full terms of all abbreviations were added in all figure legends and table footnotes. The number of replicates is n=3 and means ± SD and added in the figure legends and table footnotes.

 

- Line 194-195, 197-203: should be transferred to the discussion.

Response: We are sorry about this question. But the line 194-195 was “These suggested that Cu-stress has reduced the nitrogen assimilation efficiency of G. lemaneiformis”. This is a description of the result, we think it is appropriate in the Results section. 3.2. Content of H2O2 and flavonoids

Line 197-203 “3.2. Content of H2O2 and flavonoids H2O2 is the main component of ROS, whereas flavonoids, non-enzymatic antioxidants prevalent in plants, serve to scavenge ROS. The content of these compounds was quantified at 24, 48 and 96 h. As depicted in Figure 2, H2O2 content, increased by 57.5-82.5%, and flavonoids increased by 20.1% and 15.6% at 48 and 96 h, respectively. In our prior studies, we observed that the contents of glutathione (GSH) and ascorbate (AsA), along with the activity levels of superoxide dismutase (SOD) and ascorbate peroxidase (APX) within the antioxidant system, were also augmented by Cu (Tang et al., 2023). These results indicated that Cu-stress has promoted the production of ROS, and the antioxidant.” It is also a description of the result, we think it is appropriate in the Results section. 3.2.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Dear Authors,

 

I have carefully read your paper entitled "Physiological, transcriptome and metabolome analysis reveal of tolerance to Cu toxicity in red macroalgae Gracilariopsis lemaneiformis" presented by Xiaojiao and cols. I found the work interesting, an example of a multidisciplinary approach (biochemical, genomic, functional) to the study of the effect of chemical substances. In general the study is correct but there are some methodological flaws that should be clarified and improved before accepting the work for publication in IJMS:

 

The citations do not follow the format established by the journal.

 

Line50-58- Indicate in full each abbreviation the first time it appears in the text.

 

Line 77. What does 981 mean

Line 82. Indicate the units in which salinity is measured

Line 91. Indicate the abbreviation for PBS

 

The Figures are fuzzy, I don't understand anything at all, especially fig.4.

 

About section 2.2. Photosynthetic pigments. Please describe the method followed or cite the bibliographic source from which the procedure was obtained.

 

In section 2.1: Materials, culture conditions and treatment methods, it is indicated how the Cu concentrations (25uM) have been prepared, but it is not indicated if the solutions have been analyzed to ensure that the nominal concentrations coincide with the nominal ones.

 

In section 2.3. Chlorophyll fluorescence parameters. The bibliographic reference of the method is not indicated, if it is an own method of the authors, please describe it in greater depth.

 

Section 2.4. Nitrate reductase activities. Please indicate the bibliographic reference of the method on which the kit used is based.

 

Section 2.5. Content of H2O2 and flavonoids. Please indicate the bibliographic reference of the method on which the kit used is based.

 

Section 2.6. Total RNA extraction. Please indicate the meaning of "CK".

 

Section 2.7. Total RNA extraction. Please briefly describe the method.

 

Section 2.8 Metabolome analysis. Has the method followed been previously published? If so, please indicate the bibliographic reference.

 

Section 2.9. Statitical Anaysis. You have indicated the country of origin of the SPSS statistical package, Plese, do the same for MS Excel and Adobe Illustrator.

 

best regards

Author Response

The citations do not follow the format established by the journal.

Response: Thank you for your comments. The reference's formatting have corrected as per the journal guidelines. 

 

Line 50-58- Indicate in full each abbreviation the first time it appears in the text.

Response: Thank you for your comments. We have added the full each abbreviation the first time it appears in the revised manuscript.

 

Line 77. What does 981 mean

Response: 981 is the name of a strain of Gracilariopsis lemaneiformis.

 

Line 82. Indicate the units in which salinity is measured

Response: The salinity units is ‰ and we added it in line 377 in the revised manuscript.

 

Line 91. Indicate the abbreviation for PBS

Response: Thank you for your comments. The full name of PBS is phosphate buffered saline and we added it in line 393 in the revised manuscript.

 

The Figures are fuzzy, I don't understand anything at all, especially fig.4.

Response: We are sorry for this question. We have replaced all of pictures to make it clearer.

 

About section 2.2. Photosynthetic pigments. Please describe the method followed or cite the bibliographic source from which the procedure was obtained.

Response: Thank you for your comments. The references to the photosynthetic pigment determination methods in Section 4.2 in the revised manuscript were added.

 

In section 2.1: Materials, culture conditions and treatment methods, it is indicated how the Cu concentrations (25uM) have been prepared, but it is not indicated if the solutions have been analyzed to ensure that the nominal concentrations coincide with the nominal ones.

Response: Thank you for your comments. The final Cu concentrations of 25 uM was diluted by configuring 25 mM stock solution and we added it in line 389 in the revised manuscript.

 

In section 2.3. Chlorophyll fluorescence parameters. The bibliographic reference of the method is not indicated, if it is an own method of the authors, please describe it in greater depth.

Response: Thank you for your comments. The references to the chlorophyll fluorescence parameters determination methods in Section 4.3 in the revised manuscript were added.

 

Section 2.4. Nitrate reductase activities. Please indicate the bibliographic reference of the method on which the kit used is based.

Response: Thank you for your comments. The references to the Nitrate reductase activities determination methods in Section 4.4 in the revised manuscript were added.

 

Section 2.5. Content of H2O2 and flavonoids. Please indicate the bibliographic reference of the method on which the kit used is based.

Response: Thank you for your comments. The references to the H2O2 and flavonoids determination methods in Section 4.5 in the revised manuscript were added.

 

Section 2.6. Total RNA extraction. Please indicate the meaning of "CK".

Response: Thank you for your comments. The meaning of "CK" was control samples and we have modified it in line 439 in the revised manuscript.

 

Section 2.7. Total RNA extraction. Please briefly describe the method.

Response: Thank you for your comments. The method of total RNA extraction was briefly described in Section 4.6 in line 441-448 in the revised manuscript.

 

Section 2.8 Metabolome analysis. Has the method followed been previously published? If so, please indicate the bibliographic reference.

Response: Thank you for your comments. The method of metabolome has been previously published and we cited this bibliographic reference in Section 4.8 in line 468 in the revised manuscript..

 

Section 2.9. Statitical Anaysis. You have indicated the country of origin of the SPSS statistical package, Plese, do the same for MS Excel and Adobe Illustrator.

Response: Thank you for your comments. The country of origin of the MS Excel and Adobe Illustrator was added in Section 4.9 in line 503-504 in the revised manuscript..

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors described the response of the rhodophyte Gracilariopsis lemaneiformis on the Cu²⁺ in terms of photosynthetic activity, pigments, gene expression and metabolite profile. The data could be valuable, but there are signifficant drawbacks. I am focusing on two main points.

1. How did the authors decided that the algae were in the state of stress under the selected Cu²⁺ concentration (25 μM)? Thus the statement about the stress are not supported.

2. Photosynthetic part is weak. Please, provide the references for the equations and provide them Fv'/Fm', rETR, NPQ, photochemical quenching (qP, not Qp!). The authors did not describe calculation of the Stern-Volmer NPQ parameter presented in the Results, however mention qP and Fv'/Fm'. Why was not the main fluorescence parameter (Fv/Fm) been calulated? The parameters of registration should be indicated (photon flux densities of the actinic, measuring, and saturating light, wavelength(s), time of actinic light action. Figure 1: units of rETR are not indicated. What is NPQ-less? 

There are alslo specific comments (please, see below).

SPECIFIC COMMENTS

-The reading of the text is strongly complicated by the excessive number of abbreviations.

-l. 19, 394-395. "arachidonic acid-a primary component of the membrane-" - it has to be supported by a reference. arachidonic acid is not related to key components of cell membranes.

-Reference format in the text is wrong.

-l. 53. ASC - the abbreviation should be defined.

-l. 82. What is the salinity of 25? What are the units?

-l. 99-100. All symbols in the equations must be explained.

-The format of figure references is wrong.

-Figure 1-5: all abbreviations should be explained in the legend.

-Figure 5: the upper panel is rather a separate table.

-l. 207-212. There is only a verbal description of results. Is it really needed?

-Table 1-3. Log 2 should be log₂.

-Avoid references in rsults (subsections 3.2-3.5). It is a signifficant drawback. Also you can combine results and discussion.

-Figure 4: the quality is low. Cannot read.

-l. 361. As well as in the culture medium.

-l. 362. It is also a cofactor of plastocyanin.

-l. 408. "increase in the chloroplast electron transport chain " - what does it mean? The non-cyclic chain also has the same size (from PSII to PSI and ferredoxin). 

-What are NO₃-N, NO₂-N, and  NH₄⁺-N. I know just three main forms of inorganic N in eukaryotes: NO₃^-, NO₂^-, and NH^₄+.

-Cadmium (Cd) should be Cd and Cuprum (Cu) should be Cu, because symbols of chemical elements are not described commonly.

-l. 451. Carotenoids and fatty acids are not secondary metabolites.

-l. 459. "suggested that PUFAs are powerful effectors" - please, explaine with correspnding references. It is commonly accepted that unsaturated fatty acids are key mediators of ROS generation in the reactions of lipid peroxydation. How can they act as antioxidants?

-Discussion shouldnot inlude figure references.

-The data on 3-hydroxy-4-keto-gama-carotene are not described in the results.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

1. How did the authors decided that the algae were in the state of stress under the selected Cu2+ concentration (25 μM)? Thus the statement about the stress are not supported.

Response: Thank you for your comments. Based on our previous study (Tang et al., 2023), a Cu concentration equal to that of IC50 (the inhibitory concentration to reduce the relative growth rate (RGR) by 50%) was chosen to prevent severe growth inhibition of G. lemaneiformis. The IC50 was approximately 25 μM, as calculated by linear interpolation. Thus, 25 μM Cu (~1.6mg/L) was chosen for the subsequent experiments. And this reason of Cu2+ concentration was added in line 380-387 in the revise manuscript.

 

Tang, Y., Chen, X., Zhang, H., Sun, X., Xu, N. 2023. 1-Aminocyclopropane-1-carboxylic acid mitigates copper stress by decreasing copper transport and inducing jasmonic acid synthesis in Gracilariopsis lemaneiformis. Environ. Exp. Bot. 208, 105237-105249. https://doi.org/10.1016/j.envexpbot.2023.105237

 

 

2. Photosynthetic part is weak. Please, provide the references for the equations and provide them Fv'/Fm', rETR, NPQ, photochemical quenching (qP, not Qp!). The authors did not describe calculation of the Stern-Volmer NPQ parameter presented in the Results, however mention qP and Fv'/Fm'. Why was not the main fluorescence parameter (Fv/Fm) been calulated?The parameters of registration should be indicated(photon flux densities of the actinic, measuring, and saturating light, wavelength(s), time of actinic light action. Figure 1: units of rETR are not indicated. What is NPQ-less?

Response: Thank you for your comments. The references for the equations was provided in line in. The result of photochemical quenching (qP), actual photochemical efficiency (Fv’/Fm’) of photosystem II (PSII) and Fv/Fm was no significant difference under Cu stress in G. lemaneiformis. Thus, the description of qP and Fv'/Fm' in method section was deleted. NPQ was directly measured by the NPQ3 program of PAM AP-C100, as a measure of photoprotective thermal dissipation of excess excitation energy. The parameters of measurement has been provided in line in .

In Figure 1, the units of rETR was provided. NPQ-less has changed to NPQ.

 

There are alslo specific comments (please, see below).

 

SPECIFIC COMMENTS

 

-The reading of the text is strongly complicated by the excessive number of abbreviations.

Response: We are sorry about it. The full terms of all abbreviations were added when first appears. 

 

-l. 19, 394-395. "arachidonic acid-a primary component of the membrane-" - it has to be supported by a reference. arachidonic acid is not related to key components of cell membranes.

Response: Thank you for your comments. Arachidonic acid , a member of the C20 (20:4, n=6) polyunsaturated fatty acids (PUFAs), is primarily found in algae and mosses but not in higher plants. It plays a crucial role as a constituent of biological membranes, a precursor of prostaglandins and many other eicosanoids (Shanab et al., 2018). We have deleted the description of "arachidonic acid-a primary component of the membrane-" in the abstract. According to the reference, arachidonic acid is a constituent of biological membranes.

 

Shanab, S.M.M., Hafez, R.M., Fouad, A.S. A review on algae and plants as potential source of arachidonic acid. J. Adv. Res. 2018, 11, 3–13. https://doi.org/10.1016/j.jare.2018.03.004

 

-Reference format in the text is wrong.

Response: Thank you for your comments. The reference's formatting have corrected as per the journal guidelines. 

 

-l. 53. ASC - the abbreviation should be defined.

Response: We are sorry about this question. ASC should be AsA and AsA was defined as Ascorbate acid in line 59-60 in the revised manuscript.

 

-l. 82. What is the salinity of 25? What are the units?

Response: The salinity units is ‰ and we added it in line 377 in the revised manuscript.

 

-l. 99-100. All symbols in the equations must be explained.

Response: Thank you for your comments. Ax is the absorbance value at x nm, V is the volume of the extraction solution (mL), and FW is the fresh weight of the seaweed (g). And we have added it in line 403-404 in the revised manuscript.

 

-The format of figure references is wrong.

Response: Thank you for your comments. We have modified the format of figure references.

 

-Figure 1-5: all abbreviations should be explained in the legend.

Response: Thank you for your comments. All abbreviations in the legend have been explained in the revised manuscript.

 

-Figure 5: the upper panel is rather a separate table.

Response: Thank you for your comments. The upper panel in Figure 5 has changed to table 4 (The KEGG pathway enrichment of DEMs).

 

-l. 207-212. There is only a verbal description of results. Is it really needed?

Response: Thank you for your comments. This part is a brief description of the data obtained by transcriptome sequencing.

 

-Table 1-3. Log 2 should be log2.

Response: Thank you for your comments. Log 2 has change to be log2 in Table 1-3.

 

-Avoid references in results (subsections 3.2-3.5). It is a signifficant drawback. Also you can combine results and discussion.

Response: Thank you for your comments. We think that the references in the results is in order to better elicit relevant content and helps to understand the results.

 

-Figure 4: the quality is low. Cannot read.

Response: We are sorry for this question. We have replaced the Figure 4 to increase its quality.

 

-l. 361. As well as in the culture medium.

Response: We are sorry for we don’t understand what the question is.

 

-l. 362. It is also a cofactor of plastocyanin.

Response: Thank you for your comments. We have added it in line 275 in revised manuscript.

 

-l. 408. "increase in the chloroplast electron transport chain " - what does it mean? The non-cyclic chain also has the same size (from PSII to PSI and ferredoxin).

Response:We are sorry for this question. "increase in the chloroplast electron transport chain " should be "increase the electron flow rate of chloroplast electron transport chain ", because transcriptome results indicated a significant up-regulation of two DEGs encoding the photosynthetic electron transport chain petH (Fig. 4A). An excessive increase in the electron flow rate of chloroplast electron transport chain could potentially lead to the production of large amounts of ROS, thereby causing oxidative stress (Pinto et al., 2003).

 

Pinto, E., Teresa C.S. Sigaud-Kutner, M.A.S., Oswaldo O. K., Morse, D., Colepicolo, P. Heavy metal-induced oxidative stress in algae. J. Phycol. 2003, 39, 1008–1018. https://doi.org/10.1111/j.0022-3646.2003.02-193.x

 

-What are NO3-N, NO2-N, and  NH4+-N. I know just three main forms of inorganic N in eukaryotes: NO3-, NO2-, and NH4+.

Response: Thank you for your comments. NO3-N, NO2-N, and NH4+-N was corrected to NO3-, NO2-, and NH4+.

 

-Cadmium (Cd) should be Cd and Cuprum (Cu) should be Cu, because symbols of chemical elements are not described commonly.

Response: Thank you for your comments. While in the article [1-3] of Cu stress that the full name of Cu is copper, so we adopted copper (Cu).

 

References:

1 Rieder, G. S., Duarte, T., Delgado, C. P., Rodighiero, A., Nogara, P. A., Orian, L., Aschner, M., Dalla Corte, C. L., & Da Rocha, J. B. T. (2024). Interplay between diphenyl diselenide and copper: Impact on D. melanogaster survival, behavior, and biochemical parameters. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 109899. Advance online publication. https://doi.org/10.1016/j.cbpc.2024.109899

2 Wang, K., Li, S., Yang, Z., Chen, C., Fu, Y., Du, H., Sun, H., Li, J., Zhao, Q., & Du, C. (2023). L-type lectin receptor-like kinase OsCORK1 as an important negative regulator confers copper stress tolerance in rice. Journal of hazardous materials, 459, 132214. https://doi.org/10.1016/j.jhazmat.2023.132214

3 Wang, K., Li, S., Yang, Z., Chen, C., Fu, Y., Du, H., Sun, H., Li, J., Zhao, Q., & Du, C. (2023). L-type lectin receptor-like kinase OsCORK1 as an important negative regulator confers copper stress tolerance in rice. Journal of hazardous materials, 459, 132214. https://doi.org/10.1016/j.jhazmat.2023.132214

 

 

-l. 451. Carotenoids and fatty acids are not secondary metabolites.

Response: Thank you for your comments. Corrected.

 

-l. 459. "suggested that PUFAs are powerful effectors" - please, explaine with correspnding references. It is commonly accepted that unsaturated fatty acids are key mediators of ROS generation in the reactions of lipid peroxydation. How can they act as antioxidants?

Response: Thank you for your comments. On one hand, unsaturated fatty acids are the target of ROS attack, such as arachidonic acid. On the other hand, unsaturated fatty acids poss reductive and antioxidant properties such as EPA (C20:5, n-3) and DHA (C22:6, n-3), which exhibit anti-inflammatory and antioxidant activities and prevent breast cancer (Shanab et al., 2018). Thus, we conclude that the significant increase in EPA content induced by Cu may be related to its antioxidant properties.

 

 

Shanab, S.M.M., Hafez, R.M., Fouad, A.S. A review on algae and plants as potential source of arachidonic acid. J. Adv. Res. 2018, 11, 3–13. https://doi.org/10.1016/j.jare.2018.03.004

 

-Discussion should not inlcude figure references.

Response: Thank you for your comments. The figure references in Discussion have deleted.

 

-The data on 3-hydroxy-4-keto-gama-carotene are not described in the results.

Response: Thank you for your comments. The data on 3-hydroxy-4-keto-gama-carotene was showed in line 258-259 and Figure 5B.

 

Comments on the Quality of English Language

Moderate editing of English language required

Response: Thank you for your comments. The editing of English language has modified.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Chen et al investigated the tolerance mechanism of red macroalgae Gracilariopsis lemaneiformis under copper toxicity through physiological, transcriptome and metabolome analysis. Although the manuscript provides some information, the manuscript needs thorough revision to be published.

- First of all, the abstract needs a proper revision. The aim of the study must be clearly mentioned in the abstract.. Abstract must explain the problem, aim of your study, novelty of the study, methodology, obtained most significant results and future prospect of the study. Please rewrite the abstract accordingly.

-In the title, authors mentioned that the study is based on physiological, transcriptome and metabolome analysis. However, the introduction only discusses about antioxidant enzymes with not proper emphasis on physiological or metabolome analysis. The aim in the last part of the introduction also discusses only about the molecular analysis. This is contradictory. Please thoroughly revise the introduction focusing the mentioned points.

-The methodology part contains major deficiencies. For example, please mention for how long the algae was cultured. When it was treated with Cu?

-In the introduction, authors mentioned about antioxidant enzymes. However, in methodology, nothing has been done related to antioxidant enzymes. In the introduction, please add information related to chlorophyll pigments and related analysis under copper toxicity with proper citations.

- Please provide information on the quality of isolated RNAs. For example, their gel picture or RIN values. You can cite proper studies such as https://www.mdpi.com/2073-4395/12/10/2421 or https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1263981/full or https://www.nature.com/articles/s42003-020-01573-1 while explaining the quality of the isolated RNA. RNA quality is important for such studies. Any contamination may affect the reliability of the obtained results.

-Please briefly add the methodology of  transcriptome analysis to the manuscript. Also, provide the information of reference genome used for transcriptome sequencing.

- In the results, Tables and Figures should be self-explanatory. For example, please mention the full form of PE content in Figure 1.  

- The discussion part seems satisfactory and does not need much revision.

- Conclusion is just a short summary of results in its present form. Please elaborate by adding the future prospects of such studies.

- At last, authors mentioned ‘Taken together, our results not only provided a theoretical basis for the healthy development of aquaculture industry, but also provide a reference for the bioremediation role of G. lemaneiformis in copper-contaminated ocean.’ How? Please only add statements that are in accordance with the obtained results.

 

I do believe that the manuscript can be accepted once the authors address the mentioned points and enrich the manuscript with the crucial information. 

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

- First of all, the abstract needs a proper revision. The aim of the study must be clearly mentioned in the abstract.. Abstract must explain the problem, aim of your study, novelty of the study, methodology, obtained most significant results and future prospect of the study. Please rewrite the abstract accordingly.

Response: Thank you for your comments. The abstract has been rewrite.

-In the title, authors mentioned that the study is based on physiological, transcriptome and metabolome analysis. However, the introduction only discusses about antioxidant enzymes with not proper emphasis on physiological or metabolome analysis. The aim in the last part of the introduction also discusses only about the molecular analysis. This is contradictory. Please thoroughly revise the introduction focusing the mentioned points.

Response: Thank you for your comments. The introduction was added the proper emphasis of physiological and metabolome analysis in line 44-60 in revised manuscript.

 

-The methodology part contains major deficiencies. For example, please mention for how long the algae was cultured. When it was treated with Cu?

Response: We are sorry about this question. The sampling periods for physiological index and the expression profiles were up to 4 d and 24 h after Cu treatment, respectively.

 

-In the introduction, authors mentioned about antioxidant enzymes. However, in methodology, nothing has been done related to antioxidant enzymes. In the introduction, please add information related to chlorophyll pigments and related analysis under copper toxicity with proper citations.

Response: Thank you for your comments. Antioxidant enzymes have been measured in our previous articles (Tang et al., 2023) and we cited it in the discussion. We mainly analyzed the changes of antioxidant enzyme gene expression in this research.

Reference:

Tang, Y., Chen, X., Zhang, H., Sun, X., Xu, N. 2023. 1-Aminocyclopropane-1-carboxylic acid mitigates copper stress by de-creasing copper transport and inducing jasmonic acid synthesis in Gracilariopsis lemaneiformis. Environ. Exp. Bot. 208, 105237-105249. https://doi.org/10.1016/j.envexpbot.2023.105237

 

-Please provide information on the quality of isolated RNAs. For example, their gel picture or RIN values. You can cite proper studies such as https://www.mdpi.com/2073-4395/12/10/2421 or https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1263981/full or https://www.nature.com/articles/s42003-020-01573-1 while explaining the quality of the isolated RNA. RNA quality is important for such studies. Any contamination may affect the reliability of the obtained results.

 

Response: Thank you for your comment. The RIN values was provided in .The RNA integrity number (RIN) of all RNA sample was bigger than 7 and belong to category A (Supplementary table 2), thus RNA quality meets the requirements of transcriptome sequencing.

 

-Please briefly add the methodology of transcriptome analysis to the manuscript. Also, provide the information of reference genome used for transcriptome sequencing.

Response: Thank you for your comment. We have added the methodology of transcriptome analysis and provided the information of reference genome used for transcriptome sequencing in line 450-466 in revised manuscript.

 

- In the results, Tables and Figures should be self-explanatory. For example, please mention the full form of PE content in Figure 1.

Response:Thank you for your comments. The full name of abbreviation in Tables and Figures was added.

 

- The discussion part seems satisfactory and does not need much revision.

Response: Thank you. 

 

- Conclusion is just a short summary of results in its present form. Please elaborate by adding the future prospects of such studies.

Response: Thank you for your comment. We modified the conclusion and added the future prospects of such studies.

 

- At last, authors mentioned ‘Taken together, our results not only provided a theoretical basis for the healthy development of aquaculture industry, but also provide a reference for the bioremediation role of G. lemaneiformis in copper-contaminated ocean.’ How? Please only add statements that are in accordance with the obtained results.

Response: Thank you for your comment. We have deleted “Taken together, our results not only provided a theoretical basis for the healthy development of aquaculture industry, but also provide a reference for the bioremediation role of G. lemaneiformis in copper-contaminated ocean.” and added statements that are in accordance with the obtained results.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

-

Author Response

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

 

2. Questions for General Evaluation         Reviewer’s Evaluation

Does the introduction provide sufficient 

background and include all relevant references?     YES

  

Are all the cited references relevant to the         YES

research?

 

Is the research design appropriate?               YES

 

Are the methods adequately described?           YES

 

Are the results clearly presented?                YES  

 

Are the conclusions supported by the results?    YES

 

3. Point-by-point response to Comments and Suggestions for Authors

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors

Thank you for improving the manuscript and including my suggestions.

I believe the manuscript can be accepted as is.

Author Response

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

 

2. Questions for General Evaluation    Reviewer’s Evaluation    Response and Revisions

Does the introduction provide sufficient

background and include all relevant references?  Can be improved    Modified

  

Are all the cited references relevant to the         YES

research?

 

Is the research design appropriate?           Can be improved    Modified

Are the methods adequately described?       Can be improved    Modified

 

Are the results clearly presented?                YES  

 

Are the conclusions supported by the results?    Can be improved    Modified

 

3. Point-by-point response to Comments and Suggestions for Authors

 

Comments: Thank you for improving the manuscript and including my suggestions. I believe the manuscript can be accepted as is.

 

Response: Thank you for your comment. We wish to express our sincere gratitude for your thorough review and valuable suggestions concerning our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

I have clearly evaluated the revised text and authors' responses. Unfortunatly, authors did not make all necessary changes in the text, despite it was marked as done in the responses. Thus, the revision is still required. Below I provide original requests with authors' responses (italicised) and my additional comments (bolded). 

1. How did the authors decided that the algae were in the state of stress under the selected Cu2+ concentration (25 μM)? Thus, the statement about the stress are not supported. It is still has not been explained, which common stress criterion was used (Car/Chl, FvF/m, other ?..). Please, indicate it.

2. "Photosynthetic" part is still weak.

-Please, provide the references for the equations and provide them Fv'/Fm', rETR, NPQ, qP. - It has not been done. It is also better to provide the equations for parameters calculations.

-The authors did not describe calculation of the Stern-Volmer NPQ parameter presented in the Results, however mention qP and Fv'/Fm'. Why did you calculate qP? The data on its analysis are absent in the results.

-“The references for the equations was provided in line in. The result of photochemical quenching (qP), actual photochemical efficiency (Fv’/Fm’) of photosystem II (PSII) and Fv/Fm was no significant difference under Cu stress in G. lemaneiformis. Thus, the description of qP and Fv'/Fm' in method section was deleted.” It has not been done.  

-“NPQ was directly measured by the NPQ3 program of PAM AP-C100, as a measure of photoprotective thermal dissipation of excess excitation energy. The parameters of measurement has been provided in line in .” It has not been done. Name of the protocol is uncommon. It should be described. The parameters of registration should be indicated (photon flux densities of the actinic, measuring, and saturating light, wavelength(s), time of actinic light action.

-“In Figure 1, the units of rETR was provided. NPQ-less has changed to NPQ” It has not been done.

3. “Arachidonic acid , a member of the C20 (20:4, n=6) polyunsaturated fatty acids (PUFAs), is primarily found in algae and mosses but not in higher plants. It plays a crucial role as a constituent of biological membranes, a precursor of prostaglandins and many other eicosanoids (Shanab et al., 2018). We have deleted the description of "arachidonic acid-a primary component of the membrane-" in the abstract. According to the reference, arachidonic acid is a constituent of biological membranes” It has not been modulated through the text (l. 186-188). I checked the reference Shanab et al. (2018). There is an information about arachidonic acid as a key component of the brain phospholipid membrane, “a precursor of prostaglandins and many other eicosanoids, … an immune-supressant, and induce inflammatory responses, blood clotting and cell signalling”. It refers to animal organisms (including human), but nit the case of algae. Thus, I cannot see the data regarding its role in algal membranes. No data on its ROS scavenging activity in plants (l. 512).

4. The reference's formatting have corrected as per the journal guidelines. It has not been done. Please, clearly see the guide. In the text, references are presented as numbers in squared brackets (not authors’ surnames and years).

5. All symbols in the equations must be explained. “Thank you for your comments. Ax is the absorbance value at x nm, V is the volume of the extraction solution (mL), and FW is the fresh weight of the seaweed (g). And we have added it in line 403-404 in the revised manuscript.” The symbols Chl, Car, PC, and PE also should be explained.

6. The format of figure references is still wrong. Please, double check the guide. In the text the word ‘Fig.’ should be written in full – ‘Figure’.

7. All abbreviations should be explained in the legend. “All abbreviations in the legend have been explained in the revised manuscript.” – please, check Figure 3.

8. Table 1-3. Log 2 should be log₂. Please, see the difference: logarithm basis is a lowercase.

9. Avoid references in results (subsections 3.2-3.5). It is a significant drawback. Also, you can combine results and discussion.

10. As well as in the culture medium. “We are sorry for we don’t understand what the question is.” Most likely, you could not evaluate Cu content in the cells, but measured it in the culture medium. It is not the same as in the cell. It should be noted.

11. "increase in the chloroplast electron transport chain " - what does it mean? It has not been modulated through the text (l. 317).

12. NO₃ should be NO₃^-, NO₂ should be NO₂^- (l. 322-323).

13. "suggested that PUFAs are powerful effectors" - please, explaine with correspnding references. It is commonly accepted that unsaturated fatty acids are key mediators of ROS generation in the reactions of lipid peroxydation. How can they act as antioxidants? “On one hand, unsaturated fatty acids are the target of ROS attack, such as arachidonic acid. On the other hand, unsaturated fatty acids poss reductive and antioxidant properties such as EPA (C20:5, n-3) and DHA (C22:6, n-3), which exhibit anti-inflammatory and antioxidant activities and prevent breast cancer (Shanab et al., 2018). Thus, we conclude that the significant increase in EPA content induced by Cu may be related to its antioxidant properties.” Here again, the reference Shanab et al. is about the role of PUFA in animal cells. Moreover, there is no data on arachidonic acid effect from this work, thus, these conclusions are not supported by the results.

14. l. 22. “were up-regulated compared with the control, indicating that they may” – upregulation of the pathways does not indicate they are involved in the detoxication. They are also could bi involved in general stress response, cell reparation, etc.

15. “The data on 3-hydroxy-4-keto-gama-carotene was showed in line 258-259 and Figure 5B.” – please, add in the discussion possible mechanism of its action.

Comments on the Quality of English Language

“The editing of English language has modified.” -  Please, check: “membrane lipid arachidonic acid” – should be “arachidonic acid residues” (l. 15), “Photosynthesis pigments” should be “Photosynthetic pigments” (l. 391).

Author Response

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

 

2. Questions for General Evaluation      Reviewer’s Evaluation  Response and Revisions

Does the introduction provide sufficient 

background and include all relevant references?   Must be improved   Modified  

Are all the cited references relevant to the       Must be improved   Modified research?

Is the research design appropriate?             Must be improved   Modified

Are the methods adequately described?        Must be improved   Modified

Are the results clearly presented?               Must be improved   Modified  

Are the conclusions supported by the results?    Must be improved   Modified

 

3. Point-by-point response to Comments and Suggestions for Authors

 

I have clearly evaluated the revised text and authors' responses. Unfortunatly, authors did not make all necessary changes in the text, despite it was marked as done in the responses. Thus, the revision is still required. Below I provide original requests with authors' responses (italicised) and my additional comments (bolded).

 

Response: Thank you for your comments on our manuscript. We found that all the comments were very helpful and strengthened the manuscript. The revisions are outlined below. In our response, the line numbers and page numbers refer to those in the initial manuscript unless otherwise stated. In addition, all revisions to the the modified version of ijms-2906259-done are indicated with yellow highlight.

 

1. How did the authors decided that the algae were in the state of stress under the selected Cu2+ concentration (25 μM)? Thus, the statement about the stress are not supported.It is still has not been explained, which common stress criterion was used (Car/Chl, Fv/Fm, other ?..). Please, indicate it.

 

Response 1: Thank you for pointing this out. Zhang et al (2020) reported that plants are considered to be under stress when environmental conditions are not ideal for growth and researchers often measure stress resistance as the rate of plant growth or survival under stress relative to control conditions. The effect of stress on plant growth can be measured as a decrease in plant growth rate or as a decrease in biomass accumulation. Thus, it is reasonable to reduce the relative growth rate of Gracilariopsis lemaneiformis by 50% as a screening condition for Cu stress. And in many papers, the heavy metal treatment concentration is determined by the change of growth, for example, the tested concentrations of As(V) based on IC₁₀-IC₅₀ of As(V) on tested rapeseed biomass (Dong et al., 2022) and a Cd concentration was screening based on EC50 (the metal concentration that results in 50% reduction in growth of the algae cells) (Tao et al., 2023).

 

References:

Zhang, H., Zhao, Y., & Zhu, J. K. (2020). Thriving under Stress: How Plants Balance Growth and the Stress Response. Developmental cell, 55(5), 529–543. https://doi.org/10.1016/j.devcel.2020.10.012

Dong, R., Liu, R., Xu, Y., Liu, W., Wang, L., Liang, X., Huang, Q., & Sun, Y. (2022). Single and joint toxicity of polymethyl methacrylate microplastics and As (V) on rapeseed (Brassia campestris L.). Chemosphere, 291(Pt 3), 133066. https://doi.org/10.1016/j.chemosphere.2021.133066

Tao, Y., He, M., Chen, B., Ruan, G., Xu, P., Xia, Y., Song, G., Bi, Y., & Hu, B. (2023). Evaluation of Cd2+ stress on Synechocystis sp. PCC6803 based on single-cell elemental accumulation and algal toxicological response. Aquatic toxicology (Amsterdam, Netherlands), 258, 106499. https://doi.org/10.1016/j.aquatox.2023.106499

 

2. "Photosynthetic" part is still weak.

-Please, provide the references for the equations and provide them Fv'/Fm', rETR, NPQ, qP. -It has not been done. It is also better to provide the equations for parameters calculations.

-The authors did not describe calculation of the Stern-Volmer NPQ parameter presented in the Results, however mention qP and Fv'/Fm'. Why did you calculate qP? The data on its analysis are absent in the results.

-“The references for the equations was provided in line in. The result of photochemical quenching (qP), actual photochemical efficiency (Fv’/Fm’) of photosystem II (PSII) and Fv/Fm was no significant difference under Cu stress in G. lemaneiformis. Thus, the description of qP and Fv'/Fm' in method section was deleted.” It has not been done.  

-“NPQ was directly measured by the NPQ3 program of PAM AP-C100, as a measure of photoprotective thermal dissipation of excess excitation energy. The parameters of measurement has been provided in line in .” It has not been done. Name of the protocol is uncommon. It should be described. The parameters of registration should be indicated (photon flux densities of the actinic, measuring, and saturating light, wavelength(s), time of actinic light action.

Response 2: We wish to express our sincere gratitude for your thorough review and valuable suggestions concerning our manuscript. Regarding your request for references for the equations Fv'/Fm', rETR, NPQ, and qP, we have carefully considered your comments and have addressed them as follows:

Regarding the Fv'/Fm' equation: Fv’ / Fm’= (Fm’ - Fo’) / Fm’.This equation assesses photosynthetic efficiency, where Fm’ and Fo’ indicate maximum and minimum fluorescence under actinic light, respectively. We have referred to [Kitajima and Butler, 1975; Ralph et al., 2002], which details the equation and its context of application. And this equation and related references have added in line xx.

Regarding the rETR equation: rETR (relative electron transport rate) is a critical indicator for assessing the rate of electron transport in photosynthesis and measured as follows: 

The maximum fluorescence under actinic light (Fm’) and estimate steady-state fluorescence under actinic lights (Fs) was detected at 7 levels of actinic irradiance at 0, 10, 20, 50, 100, 300, 500 and 1000 μmol photons m⁻² s⁻¹ with 30 s intervals. Using the measured fluorescence, the PAM method makes it possible to estimate a relative electron transport rate (rETR (I); μmol electrons m⁻² s⁻¹) for each level of actinic irradiance (I; μmol photons m⁻² s⁻¹) calculated according to Van Kooten and Snel (1990) as follow:

rETR (I) = [1- Fs(I) / Fm’(I)] × I

rETR (I), Fs (I) and Fm’(I) represent relative electron transport rate, estimate steady-state fluorescence, and maximum fluorescence at I (μmol photons m⁻² s⁻¹) level of actinic light, respectively.

To estimate the photosynthetic parameters and the maximum photosynthetic capacity (rETRmax, μmol electrons m⁻² s⁻¹), the mechanistic model of Eilers and Peeters (1988) was applied, the rETR (I) was then fitted using the following equation via OriginPro 9 (OriginLab) software and rETRmax was calculated as follows [58]:

rETR (I) = I/ (aI2 + bI + c)

rETRmax = 1/ (b+2(a × c)1/2)

a, b, and c are fitting parameters, and I is the photon flux density of active light (μmol photons m-2 s-1). rETRmax represent maximum photosynthetic capacity.

Regarding the NPQ equation: NPQ = (Fm - Fm') / Fm'.

Fm indicate that the maximum level of fluorescence in response to a light saturating flash following a 15-minute dark adaptation period. Fm’ represent the maximum fluorescence under actinic light. We have cited [Maxwell and Johnson, 2000] to support the NPQ calculation method we employed.

Regarding the qP equation: qP= (Fm’ - Fs) / (Fm’ - Fo’)

Fm’, Fs and Fo’ represent the maximum, steady-state fluorescences and minimum fluorescence under actinic light, respectively.

The methodology for calculating this indicator is based on [Roháček., 2002].

The measurement parameters are as follows: photon flux densities of the actinic: 100 μmol photons m⁻² s⁻¹, measuring light: 0, 10, 20, 50, 100, 300, 500 and 1000 μmol photons m⁻² s⁻¹, and saturating light: 1000 μmol photons m⁻² s⁻¹, wavelength(s): 620 nm, time of actinic light action: 15 ms.

We have added these references to the sections 3.3. “Chlorophyll fluorescence parameters” of our manuscript, ensuring that the source of each equation is clearly annotated.

 

References:

Ralph, P.J., Short, F.T. Impact of the wasting disease pathogen, Labyrinthula zosterae, on the photobiology of eelgrass Zostera marina. Mar. Ecol. Prog. Ser. 2002, 226, 265–271. https://doi.org/10.1371/journal.pone.0062169

Genty, B., Briantais, J.M., Baker, N.R. The relationship between the quantum yield of photosynthesis electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta. 1989, 990, 87–92. https://doi.org/10.1016/S0304-4165(89)80016-9

Roháček, K. Chlorophyll Fluorescence Parameters: The Definitions, Photosynthetic Meaning, and Mutual Relationships. Pho-tosynthetica. 2002, 40, 13–29. https://doi.org/10.1023/A:1020125719386

Maxwell, K., Johnson, G.N. Chlorophyll fluorescence—a practical guide. J. Exp. Bot. 2000, 51 (345), 659–668. https://doi.org/10.1093/jexbot/51.345.659.

 

 

-“In Figure 1, the units of rETR was provided. NPQ-less has changed to NPQ” It has not been done.

 

Response: We are sorry that we did not upload the modified Figure 1 in last version. The units of rETR was provided and NPQ-less has changed to NPQ in modified Figure 1 and this has been uploaded in revised manuscript.

 

 

3. “Arachidonic acid , a member of the C20 (20:4, n=6) polyunsaturated fatty acids (PUFAs), is primarily found in algae and mosses but not in higher plants. It plays a crucial role as a constituent of biological membranes, a precursor of prostaglandins and many other eicosanoids (Shanab et al., 2018). We have deleted the description of "arachidonic acid-a primary component of the membrane-" in the abstract. According to the reference, arachidonic acid is a constituent of biological membranes” It has not been modulated through the text (l. 186-188). I checked the reference Shanab et al. (2018). There is an information about arachidonic acid as a key component of the brain phospholipid membrane, “a precursor of prostaglandins and many other eicosanoids, … an immune-supressant, and induce inflammatory responses, blood clotting and cell signalling”. It refers to animal organisms (including human), but nit the case of algae. Thus, I cannot see the data regarding its role in algal membranes. No data on its ROS scavenging activity in plants (l. 512).

 

Response 3: We apologize for not looking at the specific literature to this quote “It plays a crucial role as a constituent of biological membranes, a precursor of prostaglandins and many other eicosanoids (Shanab et al., 2018).” We have reviewed the relevant literature on arachidonic acid in algae, which is mainly about the content, biosynthesis and metabolism pathway in algae, but there is no study on its subcellular localization. Thus, we deleted the description about that “ arachidonic acid is a constituent of biological membranes” in abstract and result section.

ROS scavenging activity of PUFA. Due to the double bond in the UFAs, UFAs are vulnerable to reactive oxygen species (ROS), which are produced in response to stress and high concentration of ROS can result in DNA damage and cell death. He and Ding (2020) reported that in plant, the chemical nature also renders C18 UFAs intrinsic antioxidants for they can directly react with and thus consume ROS. Othman et al (2024) found that during Ganoderma boninense infection of oil palm, one of the most enriched pathways in the partial resistant progeny was the biosynthesis of unsaturated fatty acids (UFAs) and they provide point of view that UFAs can help the oil palm deal with the infection related stress and turn into ROS scavenger as they can directly react with ROS and consume it. In planta, C18 UFAs are utilized as raw material to produce numerous aliphatic compounds, including membrane glycerolipids, TAG, cutin/suberin, jasmonates, and nitroalkenes (NO2-FAs). It is remarkable that all these products, as well as C18 UFAs themselves, take part in plant defense against various biotic and abiotic stresses (He and Ding, 2020). Similarly, in algae, Ritter et al. (2008) reported the synthesis of octadecanoid and eicosanoid oxygenated derivatives in Laminaria digitata following the exposure to Cu stress. The levels of PUFA C18:2 (n−6), C18:3 (n−3) and C20:4 (n−6) was enhanced under salt stress (Kumar et al., 2010) and that of C20:4 (n-6) and C20:3 (n-6) was increased in response to desiccation induced oxidative stress (Kumar et al., 2011), suggesting that PUFA might deal with the abiotic stress in algae. Thus, it can be inferred that PUFAs could also be the robust effectors for scavenging the ROS in algae.

 

References:

Ritter, A., Goulitquer, S., Salaun, J., Tonon, T., Correa, J.A., Potin, P., 2008. Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in

the brown algal kelp Laminaria digitata. New Phytol. 180, 809–821.

Kumar, M., Kumari, P., Gupta, V., Reddy, C. R. K., Jha, B. Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to salinity induced oxidative stress. J. Exp. Mar. Biol. Ecol. 2010, 391(1-2), 27-34. https://doi.org/10.1016/j.jembe.2010.06.001

Kumar, M., Gupta, V., Trivedi, N., Kumari, P., Bijo, A.J., Reddy, C.R.K., Jha, B. Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta). Environ. Exp. Bot. 2011, 72 (2), 194-201. https://doi.org/10.1016/j.envexpbot.2011.03.007

Ohlrogge, J., and Browse, J. (1995). Lipid biosynthesis. Plant Cell 7, 957–970. doi: 10.1105/tpc.7.7.957

He, M., He, C. Q., and Ding, N. Z. (2018). Abiotic stresses: general defenses of land plants and chances for engineering multistress tolerance. Front. Plant Sci. 9:1771. doi: 10.3389/fpls.2018.01771

He, M., & Ding, N. Z. (2020). Plant Unsaturated Fatty Acids: Multiple Roles in Stress Response. Frontiers in plant science, 11, 562785. https://doi.org/10.3389/fpls.2020.562785

Othman, A., Lau, BYC., Nurazah, Z., Shahwan, S. et al. (2024).Comparative proteomic and metabolomic studies between partial resistant and susceptible oil palm reveal the molecular mechanism associated with Ganoderma boninense infection, Physiological and Molecular Plant Pathology,129, 102198. https://doi.org/10.1016/j.pmpp.2023.102198

 

 

 

4. The reference's formatting have corrected as per the journal guidelines. It has not been done.Please, clearly see the guide. In the text, references are presented as numbers in squared brackets (not authors’ surnames and years).

Response: 

Response 4: Thank you for comments, the reference's formatting have corrected. 

 

5. All symbols in the equations must be explained. “Thank you for your comments. Ax is the absorbance value at x nm, V is the volume of the extraction solution (mL), and FW is the fresh weight of the seaweed (g). And we have added it in line 403-404 in the revised manuscript.” The symbols Chl, Car, PC, and PE also should be explained.

Response 5: The symbols Chl, Car, PC, and PE has been explained in revised manuscript.

 

 

6. The format of figure references is stillPlease, double check the guide. In the text the word ‘Fig.’ should be written in full – ‘Figure’.

Response 6: Thank you for comments. Corrected.

 

7. All abbreviations should be explained in the legend. “All abbreviations in the legend have been explained in the revised manuscript.” – please, check Figure 3.

Response 7: The abbreviations of genes involved in the pathway in figure 3 has been added in the legend in revised manuscript. The explain of these abbreviations as follow:

GENE, LTA4H: gene encoding leukotriene-A4 hydrolase, EPHX2: genes encoding soluble epoxide hydrolase, HPGDS: gene encoding prostaglandin-H2 D-isomerase, DEGS: gene encoding sphingolipid 4-desaturase, galA: gene encoding alpha-galactosidase, DOPA: gene encoding dihydroxyphenylalanine. Metabolites, LTA4: leukotriene-A4, LT B4: leukotriene-B4, EET: Epoxyeicosa-5.8.11-trienoic acid, DHET: Dihydroxyeicosa-5,8,11-trienoic acid, PGH2: Prostaglandin H2, PGD2: Prostaglandin D2. L-DOPA: dihydroxyphenylalanine.

 

 

8. Table 1-3. Log 2 should be log2.Please, see the difference: logarithm basis is a lowercase.

Response 8: Corrected. All of Log 2 has been modified to log2 in whole manuscript.

 

9. Avoid references in results (subsections 3.2-3.5). It is a significant drawback. Also, you can combine results and discussion.

Response 9: Thank you for your suggestion. We have combined the results and discussion section.

 

10. As well as in the culture medium. “We are sorry for we don’t understand what the question is.” Most likely, you could not evaluate Cu content in the cells, but measured it in the culture medium. It is not the same as in the cell. It should be noted.

Response 10: We are sorry about it. The volume of Cu stock was added to the medium at the beginning was the same, and the Cu content in the medium was not measured.

 

11. "increase in the chloroplast electron transport chain " - what does it mean?It has not been modulated through the text (l. 317).

Response 11: We have deleted the sentence “increase in the chloroplast electron transport chain”, because this conclusion is not too rigorous due to we found that the maximum photosynthetic electron transfer rate was significantly down-regulated after Cu stress in chlorophyll fluorescence parameters

 

12. NO3should be NO3-, NO2 should be NO2- (l. 322-323).

Response 12: Thank you for your suggestion. Corrected.

 

13. "suggested that PUFAs are powerful effectors" - please, explaine with correspnding references. It is commonly accepted that unsaturated fatty acids are key mediators of ROS generation in the reactions of lipid peroxydation. How can they act as antioxidants? “On one hand,unsaturated fatty acids are the target of ROS attack, such as arachidonic acid. On the other hand, unsaturated fatty acids poss reductive and antioxidant properties such as EPA (C20:5, n-3) and DHA (C22:6, n-3), which exhibit anti-inflammatory and antioxidant activities and prevent breast cancer (Shanab et al., 2018). Thus, we conclude that the significant increase in EPA content induced by Cu may be related to its antioxidant properties.” Here again, the reference Shanab et al. is about the role of PUFA in animal cells. Moreover, there is no data on arachidonic acid effect from this work, thus, these conclusions are not supported by the results.

 

Response 13: Thank you for your comments. Due to the double bond in the UFAs, UFAs are vulnerable to reactive oxygen species (ROS), which are produced in response to stress and high concentration of ROS can result in DNA damage and cell death. He and Ding (2020) reported that in plant, the chemical nature also renders C18 UFAs intrinsic antioxidants for they can directly react with and thus consume ROS. Othman et al (2024) found that during Ganoderma boninense infection of oil palm, one of the most enriched pathways in the partial resistant progeny was the biosynthesis of unsaturated fatty acids (UFAs) and they provide point of view that UFAs can help the oil palm deal with the infection related stress and turn into ROS scavenger as they can directly react with ROS and consume it. In planta, C18 UFAs are utilized as raw material to produce numerous aliphatic compounds, including membrane glycerolipids, TAG, cutin/suberin, jasmonates, and nitroalkenes (NO2-FAs). It is remarkable that all these products, as well as C18 UFAs themselves, take part in plant defense against various biotic and abiotic stresses (He and Ding, 2020). Similarly, in algae, the levels of PUFA C18:2 (n−6), C18:3 (n−3) and C20:4 (n−6) was enhanced under salt stress (Kumar et al., 2010) and that of C20:4 (n-6) and C20:3 (n-6) was increased in response to desiccation induced oxidative stress (Kumar et al., 2011), suggesting that PUFA might deal with the abiotic stress in algae.

The most prominent theme in red algal arachidonic acid biosynthesis is the metabolism of C20 PUFAs via a 12-LOX activity. Besides the primary hydroperoxy fatty acid products, among the secondary products that have been detected are hydroxy fatty acids, diols, epoxy fatty acids, prostaglandins, and leukotrienes (28). Marine red alga Chondrus crispus (C. crispus) produced C20 PUFAs and activated the metabolism of ARA by fatty acid oxidases to generate hydroperoxides and cyclopentenones such as prostaglandins when challenged by pathogen extracts. And the 12-HPETE of C20 hydroperoxides conferred an induced resistance to the diploid phase, while treated with PGA1-2 and PGD1-2 did not effect, indicates that the role of most of the oxidative metabolites of ARA in algae is unknown or not necessarily effective. In this study, the expression of genes induced by Cu stress encoding enzymes that oxidize polyunsaturated fatty acids such as leukotriene-A4 hydrolase (LTA4H), prostaglandin-H2 D-isomerase (HPGDS) and soluble epoxide hydrolase (EPHX2), which catalyze ARA to leukotriene-B4, dihydroxyeicosa-5,8,11-trienoic acid and prostaglandins 2 , respectively. These results showed that an excess of Cu led to the enzymatic oxidation of ARA, resulting in the formation of lipid peroxidation products in G. lemaneiformis.

 

References:

He, M., Ding, N. Z. (2020). Plant Unsaturated Fatty Acids: Multiple Roles in Stress Response. Frontiers in plant science, 11, 562785. https://doi.org/10.3389/fpls.2020.562785

Othman, A., Lau, BYC., Nurazah, Z., Shahwan, S. et al. (2024).Comparative proteomic and metabolomic studies between partial resistant and susceptible oil palm reveal the molecular mechanism associated with Ganoderma boninense infection, Physiological and Molecular Plant Pathology,129, 102198. https://doi.org/10.1016/j.pmpp.2023.102198

Kumar, M., Kumari, P., Gupta, V., Reddy, C. R. K., Jha, B. Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to salinity induced oxidative stress. J. Exp. Mar. Biol. Ecol. 2010, 391(1-2), 27-34. https://doi.org/10.1016/j.jembe.2010.06.001

Kumar, M., Gupta, V., Trivedi, N., Kumari, P., Bijo, A.J., Reddy, C.R.K., Jha, B. Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta). Environ. Exp. Bot. 2011, 72 (2), 194-201. https://doi.org/10.1016/j.envexpbot.2011.03.007

Bouarab, K., Adas, F., Gaquerel, E., Kloareg, B., Salaün, J. P., & Potin, P. (2004). The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways. Plant physiology, 135(3), 1838–1848. https://doi.org/10.1104/pp.103.037622

 

14. 22. “were up-regulated compared with the control, indicating that they may” – upregulation of the pathways does not indicate they are involved in the detoxication. They are also could bi involved in general stress response, cell reparation, etc.

Response: Thank you for your suggestion. We modified to “Additionally, the metabolite such as flavonoids, 3-O-methylgallic acid, 3-hydroxy-4-keto-gama-carotene, and eicosapentaenoic acid were up-regulated compared with the control, indicating that they might play roles in response to Cu stress.”

 

15. “The data on 3-hydroxy-4-keto-gama-carotene was showed in line 258-259 and Figure 5B.” – please, add in the discussion possible mechanism of its action.

Response 15: Thank you for your suggestion. The isolated product 3-hydroxy-4-keto-gama-carotene showed potent singlet oxygen (¹O₂), one kind of ROS, quenching activity (Maoka et al., 2012). And we have added this reference in line in revised manuscript.

 

Maoka, T., Etoh, T., Osawa, A., & Shindo, K. (2012). Characterization and singlet oxygen quenching activity of (3R)-3-hydroxy-4-ketotorulene and (3R)-3-hydroxy-4-keto-γ-carotene isolated from the yeast Xanthophyllomyces dendrorhrous. Journal of oleo science, 61(7), 401–406. https://doi.org/10.5650/jos.61.401

 

 

4. Comments on the Quality of English Language

“The editing of English language has modified.”-Please, check: “membrane lipid arachidonic acid” – should be “arachidonic acid residues” (l. 15), “Photosynthesis pigments” should be “Photosynthetic pigments” (l. 391).

Response : Thank you for your suggestion. The “membrane lipid arachidonic acid” has been changed to “arachidonic acid residues” in line 15 in revised manuscript. The “Photosynthesis pigments” has been modified to “Photosynthetic pigments” in line 381 in revised manuscript.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Please thoroughly revise the manuscript as per the previously given suggestions.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Response to Reviewer 4 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted in the re-submitted files.

 

2. Questions for General Evaluation    Reviewer’s Evaluation    Response and Revisions

Does the introduction provide sufficient

background and include all relevant references?  Must be improved  Modified

  

Are all the cited references relevant to the         Must be improved  Modified

research?

Is the research design appropriate?          Must be improved  Modified

Are the methods adequately described?       Must be improved  Modified

Are the results clearly presented?               Must be improved  Modified

Are the conclusions supported by the results?    Must be improved  Modified

 

3. Point-by-point response to Comments and Suggestions for Authors

 

Comments: Please thoroughly revise the manuscript as per the previously given suggestions

 

Response: Thank you for your comment. We wish to express our sincere gratitude for your thorough review and valuable suggestions concerning our manuscript.We have made a complete revision of the manuscript based on your previously given suggestions and all revisions to the the modified version of ijms-2906259-done are indicated with yellow highlight.

 

4. Comments on the Quality of English Language

Moderate editing of English language required

 

Response: Thank you for your comment. Modified.