Physiological and Transcriptomic Analyses Reveal Commonalities and Specificities in Wheat in Response to Aluminum and Manganese

Round 1

Reviewer 1 Report

The subject of the manuscript is consistent with the scope of the Journal. The manuscript is interesting.

The abstract faithfully conveys the scope of investigations and conclusions drawn. The keywords are appropriate to the scope of the research. This study integrated physiological and transcriptome analysis to reveal the specificity and commonality in the toxicity and tolerance mechanisms of wheat under Al and Mn stresses, providing a new perspective on the molecular network of plants responding to multiple stresses in acid soil and further discovering candidate genes for wheat Al and Mn tolerance, providing genetic resources.

I think the paper is quite good. It needs some corrections:

1) the research hypotheses and aims are not clearly formulated,

2) how was SPAD measured on poorly developed plants after such a short vegetation period?

3) how did you dry and grind plant materials?

4) add the name of the apparatus and the method of digestion you use for the analysis of macro- and microelements in plants,

5) add information about analytical quality control (CRM, standards, etc.) for Al, Mn, Ca, Mg, Zn, and Fe concentrations in plants to Materials and Methods section,

6) why you used p ≤ 0.05 for statistical analyses of results from laboratory experiment?

7) add references to statistical methods,

8) indicate possible practical applications of the research results,

9) standardise References section,

10) format all sections of the manuscript according to Instructions for Authors.

Other sections are quite good.

You need to check your manuscript very carefully and correct any errors and complete any missing data.

Author Response

1. the research hypotheses and aims are not clearly formulated

Hypothesis: Due to differences between Al and Mn in the binding site, distribution, and bio-logical functions in plants, some plants’ responses (toxicity and resistance) to Al and Mn are specific or similar but play a different role and are dominated by various genes. (Line 69-71, highlighted).

Aims: This study integrated physiological and transcriptome analysis to reveal the specificity and commonality in the toxicity and tolerance mechanisms of wheat under Al and Mn stresses, providing a new perspective on the molecular network of plants responding to multiple stresses in acid soil and further discovering candidate genes for wheat Al and Mn tolerance, providing genetic resources. (Line 107-111, highlighted).

2. how was SPAD measured on poorly developed plants after such a short vegetation period?

The measured leaf area of SPAD-502 is 2 mm×3 mm. Although the wheat leaf in our experiment is relatively narrow, it can still completely cover the sensor.

3. how did you dry and grind plant materials?

The plant materials were dried in an oven at 105°C for 1 hour, followed by drying at 80°C for 3 hours until a constant weight was achieved. Then, they were ground using a JC-FW100 grinder. This has been added into the article (Line 145-147).

4. add the name of the apparatus and the method of digestion you use for the analysis of macro- and microelements in plants,

It was revised according to the reviewer’s suggestion (Line 149-152).

5. add information about analytical quality control (CRM, standards, etc.) for Al, Mn, Ca, Mg, Zn, and Fe concentrations in plants to Materials and Methods section,

It was revised according to the reviewer’s suggestion (Line 155-156).

The mixture of standards was prepared using certified reference materials (CRM) purchased from Sigma-Aldrich for Al, Mn, Ca, Mg, Zn, and Fe determination.

6. why you used p≤0.05 for statistical analyses of results from laboratory experiment?

The biological samples were collected from different batches and barrels. Treatment differences were analyzed using a conventional level of p<0.05, accounting for errors among biological replicates.

7. add references to statistical methods

It was revised according to the reviewer’s instruction (Line 124 and references thereafter).

8. indicate possible practical applications of the research results

The practical applications of the research results span various aspects of agriculture, including crop breeding, soil management, nutrient optimization, and the development of stress-tolerant crop varieties, ultimately contributing to improved crop productivity and sustainability in areas affected by Al and Mn toxicity in acid soils.

9. standardize References section

According to the reviewer's instructions, we revised the document. We used EndNote to prepare the references and formatted the document based on MDPI output style.

10. format all sections of the manuscript according to Instructions for Authors.

According to the reviewer's instructions, we revised the manuscript using the cimb-template.docx and following the Instructions for Authors.

Reviewer 2 Report

All my observations are highlighted in the text. These observations are to aid the study.

Comments for author File: Comments.pdf

Authors need to refine their English before publication.

Author Response

1. Based on the reviewer's instructions labeled and highlighted in the text, we have made all the necessary revisions to the text and formatting (highlighted in yellow background). We appreciate the valuable suggestions and convenience provided by the reviewer and have addressed them accordingly. However, we have retained some specialized terms and fixed expressions used by previous researchers, such as Solute carrier family 40 member I; XTH: Glycan synthases and glycosyltransferases; NAC: NAM, ATAF1/2, and cup-shaped cotyledon-2.

2. We substitute a couple of keywords not included in the title. [Line 33]

3. We add pertinent citation for the first paragraph of section Introduction (Line 38, Line 44).

4. We used standard units and formats (highlighted in yellow background).

5. Software and online analytical platforms produced heatmaps (Figure 6~12, especially Figure 7 and Figure 10); they did not provide enough options for color or pattern, and thus, we cannot choose other color or patterns in white and black to distinguish the represented items.

6. Every target node and line information can be displayed separately and highlighted in real-time in software; however, only the overall information of all nodes and lines will be simultaneously displayed after being exported as a tiff bitmap file (Figure 11). We have adjusted the parameter threshold to display the most critical nodes and lines. Alternatively, we also provided vector-images in PDF format for editors to improve the clarity of the image when necessary.

7. The Gene description column in Table 1 describes the gene-encoded protein, function, or metabolic pathways rather than the gene itself. It does not need to be in italics.