Nitrate Reductase Genes AtNIA1 and AtNIA2 Confer Heat Stress Resilience via ROS Homeostasis and HSP Expression in Arabidopsis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Nitrate Reductase Genes AtNIAl and AtNIA2 Confer Heat Stress Resilience via ROS Homeostasis and HSP Expression in Arabidopsis

Nusrat Jahan Methela, Mohammad Shafiqul Islam, Mahir Faysal, Moon-Sub Lee, Byung-Wook Yun and Bong-Gyu Mun

Nitrate reductase is a key enzyme in nitrate metabolism. It is responsible for the uptake of nitrate compounds and overall nitrogen metabolism. Another important role of this enzyme is its possible involvement in the formation of nitrogen monoxide. Plant nitrate reductase is capable of reducing nitrite to NO, a process known as "nitrite:NO reductase activity."

The authors hypothesized that AtNIA1 and AtNIA2 may contribute differentially to plant thermal defenses via NO signaling.

To study heat stress tolerance, successful models were selected: the wild-type Arabidopsis thaliana ecotype Columbia and its mutant lines for nitrate reductase enzymes. Comparing the responsiveness of the wild-type and mutant lines allows one to assess sensitivity to stress responses induced by high temperatures, as well as the activation of defense mechanisms.

In the experiment, the authors allowed the plants time to recover after exposure to high temperatures. The experiment simulated a possible natural situation where daytime heat alternates with cooler hours at night. Despite this, the authors found more pronounced physiological damage in the mutant lines, indicating the involvement of enzymes in the plant's heat tolerance response. Furthermore, they demonstrated differential regulation of antioxidant enzymes in response to elevated temperatures: increased catalase activity in mutants with the AtNIA1 gene knockout and ascorbate peroxidase activity in the AtNIA2 gene knockout mutant.

In addition to changes in metabolite levels and enzyme activity, the authors found differences in changes in the expression of heat shock protein and transcription factor genes.

The obtained results allow us to approach the deciphering of the mechanisms that switch normal metabolism to protective reactions.

 

Comments:

 

We agree that the article title and the names of the genes studied imply the object of study, but the Latin name of the object of study should be included in both the Abstract and the Keywords.

The Columbia ecotype of Arabidopsis should be specified in the Methodology, not just its abbreviation, Col.

Please clarify under the figures or in the Methodology how the different lines are compared: within control conditions and under the conditions of the active factor, or an end-to-end comparison.

 

The authors attribute the phenotypic changes to changes in plant color, but Figure 2 clearly shows differences in the sizes of wild-type plants and various mutants. The authors are also asked to note this fact. The AtNIA1 knockout mutant was smaller, suggesting that the disturbances are related to the coordination of nitrogen nutrition and, consequently, growth processes.

Please separate the caption to Figure 4 from the text of the manuscript.

The authors' interpretation of the significant increase in AtMBFlc expression after 3 and 6 hours in the AtNIA2 knockout mutant is unclear.

Comments for author File: Comments.pdf

Author Response

We have carefully revised the manuscript in response to the reviewers’ valuable comments. Their constructive feedback has significantly helped improve the overall quality and organization of the manuscript. We have incorporated the suggested revisions to the fullest extent possible throughout the manuscript.

Our detailed, point-by-point responses to the reviewers’ comments are provided in a separate Word file. The updated portions in the revised manuscript have been highlighted in yellow for ease of reference.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors studied the role of the AtNIA1 and AtNIA2 genes, encoding nitrate reductase, in regulating plant tolerance to heat stress. A. thaliana mutants atnia1 and atnia2 were less tolerant of elevated temperatures than Col-0, as evidenced by water loss, reduced chlorophyll levels, and a number of other physiological parameters. MDA and H2O2 accumulated in higher quantities in the mutants than in Col-0. The expression of genes involved in plant responses to heat stress was studied. It is concluded that nitrate reductase-dependent metabolic pathways are involved in plant tolerance to elevated temperatures by regulating water balance, membrane stability, ROS detoxification, and other factors.

The experimental work performed was fairly clean. However, the reviewer believes that additional experiments would be useful to make the obtained results more substantiated and compelling.

1. Why didn't the authors use a double mutant for both nitrate reductase genes? Without analysis of the double mutant, the work seems incomplete.
2. In the manuscript, the authors repeatedly refer to NO levels, emphasizing its important role in heat stress tolerance. Why didn't the authors determine NO levels in their experiments?
3. It would be desirable to provide the original data on which Figure 5 was based in Suppl. materials so that the numerical values of transcript levels could be compared, if necessary.
4. The MDA determination method used by the authors is used worldwide; however, it is not specific enough. It determines not only MDA, but also a mixture of compounds that interact with thiobarbituric acid (TBARS). For animal tissues, the use of 1-methyl-2-phenylindole increases the specificity of the reaction. In all cases involving plants, it is recommended to refer to the TBARS determination rather than MDA (Johnston et al. 2007. Plant Physiol. Bioch. 45, 108–112. DOI: 10.1016/j.plaphy.2007.01.011). This does not change the essence of the result obtained, but makes its interpretation more accurate.
5. When describing centrifugation conditions, rpm and time are usually specified. This is insufficient, since acceleration may vary depending on the centrifuge and rotor. It is necessary to specify the centrifuge type and rotor type, or indicate the g value.
6. The HSP17.4 protein is referred to differently in the text and in the figures: HSP17.4 or HSP17. This designation should be standardized.
7. “The suppressed transcriptional activation of these genes…” Transcriptional activation of genes has not been studied. Transcript levels of individual genes were studied. Transcription activation is studied using other methods.
8. "Protein expression" is a slang term, although it is sometimes used. “Gene expression” is usually used.
9. In section "2.5. Statistical Analysis," indicate the number of biological replicates.
10. Specify the light source and light intensity used to grow A. thaliana plants.

Author Response

We have carefully revised the manuscript in response to the reviewers’ valuable comments. Their constructive feedback has significantly helped improve the overall quality and organization of the manuscript. We have incorporated the suggested revisions to the fullest extent possible throughout the manuscript.

Our detailed, point-by-point responses to the reviewers’ comments are provided in a separate Word file. The updated portions in the revised manuscript have been highlighted in yellow for ease of reference.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This paper investigates the functional differences of the nitrate reductase genes AtNIA1 and AtNIA2 in Arabidopsis thaliana in terms of heat stress tolerance, and systematically explores their regulatory mechanisms in reactive oxygen species balance and heat shock protein expression. The research design is reasonable, the experimental methods are appropriate, and the data analysis is thorough, providing a new perspective for understanding the heat adaptation mechanism of plants and having good scientific value. The manuscript structure is clear, but there are some issues in language expression, format details, and figure layout. It is recommended to make corrections before publication.

1. The keyword "mutant" is suggested to be changed to "Arabidopsis mutants" for more specificity.
2. In many places in the text, sentences are connected with ";", which is suggested to be changed to commas or the sentence structure be adjusted.
3. Both "atnia1" and "nia1", "atnia2" and "nia2" appear in the text, with inconsistent expressions and font formats. It is recommended to correct them throughout the text.
4. "knock-out" and "knockout", "wild type" and "wild-type" are used interchangeably in the text. It is recommended to unify them as "knockout" and "wild-type".
5. There are some grammatical errors and inconsistent expressions in the text (e.g., "loss of AtNIA1 plants" should be changed to "AtNIA1 knockout plants", "AP atity" is a spelling mistake for "APX activity"). A thorough review and correction are needed.
6. In section 3.3 of the results, the abbreviations and full names of antioxidant enzyme activities are mixed. It is recommended to correct them.
7. The contents of Figures 4 and 5 in the results section are repetitive. It is suggested to merge them.
8. It is recommended to reformat all figures to make their layout neater and more aesthetically pleasing.
9. The mechanism model in Figure 6 should have clear annotations of arrow directions and the action nodes of key signaling molecules to make the model clearer.
10. The format of the references needs to be adjusted and unified. The "https://" prefix is missing in the DOI format of some references. There are a large number of repeated letter sequences in reference 25 on page 12, which is obviously a layout error and needs to be corrected.

Author Response

We have carefully revised the manuscript in response to the reviewers’ valuable comments. Their constructive feedback has significantly helped improve the overall quality and organization of the manuscript. We have incorporated the suggested revisions to the fullest extent possible throughout the manuscript.

Our detailed, point-by-point responses to the reviewers’ comments are provided in a separate Word file. The updated portions in the revised manuscript have been highlighted in yellow for ease of reference.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Methela and co-authors deals with plant thermotolerance and nitrate reductase-dependent signaling, combining physiological, biochemical and transcriptional analyses. The authors use Arabidopsis knockout mutants and provides novel information into the different roles of AtNIA1 and AtNIA2 during heat stress. However, before the manuscript can be considered for publication, a few things need to be improved:

1. A major issue is that the manuscript presents NO signaling as the primary mechanism underlying the reported phenotypes, although there is an absence of actual assessment of NO levels or nitrate reductase activity. There is no biochemical proof for NO production via typical detection methods (DAF-FM fluorescence, chemiluminescence or fluorometric tests). Without this data, the proposed mechanism is not adequately supported.

2. The interpretation of physiological and transcriptional data in the Discussion should be clarified. The mutants atnia1 and atnia2 show increased heat sensitivity, but the late induction of MBF1c in atnia2 is discussed as a potential compensatory response. The authors should clarify if this is a delayed stress-signaling mechanism or improved thermotolerance, and if the two NR isoforms act at different stages or via different regulatory pathways.

3. The Discussion emphasizes ABA signaling pathways but ABA levels were not measured or ABA marker genes or treatments are not included. Thus, current data do not clearly support this link. The authors should present ABA-related interpretations based on existing literature or reduce the emphasis on ABA as a key mechanism.

4. Most experiments use only three biological replicates, which is minimal given the complexity of the experimental design. Also, multiple statistical tests are applied without clear justification, and Duncan’s multiple range test is used despite its relatively high error rate. The authors should provide justification for the statistical approach, consider using two-way ANOVA where appropriate.

5. The Results section contains several issues that compromise readability. Section 3.1 includes an entire sentence that appears twice consecutively without modification (describes the reduction in relative water content and chlorophyll content in the mutants). Also, there are too detailed descriptions that simply restate what is already visible in the figures. They should focus on statistically significant differences and unexpected patterns not just narrating what readers can see by themselves. The highest redundancy is the gene expression data in Section 3.4. The same dataset appears in two formats - individual bar plots (Figure 4) and heatmaps (Figure 5) as separate textual descriptions of each is provided. The results needs rigorous editing before publication.

6. Phenotypic observations presented in Fig. 2 lack quantitative support. The visual differences in leaf yellowing between Col-0 and the mutants are seen, but there are no measurements to support these observations. Basic quantitative data such as rosette diameter, leaf area, total fresh or dry biomass or even quantified color analysis would transform this from a qualitative observation into experimental  evidence. The figure legend states only “Phenotypic view of the genotypes under heat stress for 5 days” without any statistical analysis or measurement data.

7. The choice and stability of Actin2 as a reference gene under heat stress are not validated. It is said that AtACT2 is the internal reference gene but it is not shown that it is stable under heat stress. This stress is known to affect the expression of housekeeping genes and validation using stable Ct values or alternative reference genes (e.g., UBQ10, PP2A) is needed.

Minors

- Grammatical and typographical errors are present (e.g., duplicated punctuation; chemical formulas are inconsistently formatted - e.g., H₂O₂ appears with proper subscripts in some sections but as H2O2 without subscripts in others.

- Gene naming should be standardized throughout (e.g., AtNIA1 vs atnia1).

- Formatting of gene and protein names should be more consistent. The transcription factor MBF1c is frequently italicised, but gene names and mutants often lack italics. This error appears consistently in the Results and Discussion sections and creates confusion if gene expression or protein function is being discussed. 

Author Response

We have carefully revised the manuscript in response to the reviewers’ valuable comments. Their constructive feedback has significantly helped improve the overall quality and organization of the manuscript. We have incorporated the suggested revisions to the fullest extent possible throughout the manuscript.

Our detailed, point-by-point responses to the reviewers’ comments are provided in a separate Word file. The updated portions in the revised manuscript have been highlighted in yellow for ease of reference.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have worked very diligently to revise the manuscript. They not only made all the necessary corrections to the text but also conducted additional experimental work. I have no comments on this article and can recommend its acceptance for publication in the journal.

Author Response

We sincerely thank the reviewer for the careful evaluation of our revised manuscript and for the positive comments.

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you to the authors for the careful revision of the manuscript. The revised version is substantially improved in clarity, structure and scientific interpretation. However, there are several remaining issues that should be corrected before final acceptance.

1. The author affiliation numbering is inconsistent - affiliations are numbered 1-5, but affiliation 4 has no corresponding superscript number assigned to any author. Also, Moon-Sub Lee is listed under affiliation 3 (Kyungpook National University), but his email domain is @chungbuk.ac.kr, which belongs to Chungbuk National University. 

2. The corresponding author marker points to Bong-Gyu Mun, but the “Author to whom correspondence should be addressed” line lists the email addresses of all authors.

3. Several sentences in the Introduction contain incorrectly used semicolons in place of commas (lines 42–53). Also, the last sentence of the first Introduction paragraph (ending at line 55) is missing a closing period. The authors are encouraged to perform a thorough language check of the entire manuscript.

4. The citation used to support the ABA measurement protocol in section 2.4 (reference 24) corresponds to a study on microbial melatonin production and plant metabolic function and has no relation to ABA quantification by GC-MS. This must be replaced with the correct primary methodological reference.

5. Similarly, reference 28, cited in the Discussion to support the statement that high temperatures induce excessive ROS production in plants, is a study on dandelion root extract and its cytotoxic effects on breast cancer cells. This citation is entirely unrelated to plant heat stress or ROS biology.

6. Section 2.5 states that PCR amplification was performed for 30 cycles. The authors may wish to verify whether this is accurate, as standard quantitative real-time PCR protocols typically employ 40-45 cycles. If intentional, a brief clarification or justification would be helpful for reproducibility.

7. The authors use AtPP2A as a reference gene for qRT-PCR normalisation, which is a widely accepted choice in Arabidopsis studies. Adding a brief citation supporting its stability under heat stress conditions would be a helpful addition to the methodology.

8. The Results section lacks a dedicated subsection for the ABA and SNO data presented in Figure 5. These results currently appear without a section heading, unlike all other results sections (3.1–3.4). A subsection heading should be added for consistency.

9. The elevated SNO levels observed in the atnia1 mutant, despite its proposed reduction in NO production, are not fully explained in the Discussion. A brief comment on the possible basis of this pattern would strengthen the manuscript and may be of interest to readers.

These are relatively minor points, and the manuscript can be accepted after their correction. 

Author Response

Thank you for your thoughtful comments. As in the previous review, we carefully revised the manuscript by incorporating the valuable suggestions provided by the reviewer. The revised parts have been highlighted in green. We sincerely appreciate your constructive comments again.

Author Response File: Author Response.pdf