Alginate Oligosaccharides Alleviate the Damage of Rice Leaves Caused by Acid Rain and High Temperature

Round 1

Reviewer 1 Report

Investigation of the positive effects of AOS in rice under acid rain and heat stress is an important field of plant biology. I think the manuscript is well-written. However, I think some results are duplicated. The organising of figures showing results without and with AOS treatment is not clear. Activity measurement of CAT is missing in case of AOS treatments. In addition, the main questions are: How can plants foliar uptake AOS? How can AOS effect stomata?

Other comments:

76. 76. Which are the components of antioxidant enzymes? Please describe briefly here SOD, CAT, POD…
77. 106. What is the time of sunset/rise? How long was the light/dark period?
78. 114. Why did you choose NaOH without KOH? Extra sodium can affect significantly on plants.
79. 128. Why did you apply AOS at night? Is there any light/dark-dependent effects of AOS like Salicylic acid?
80. 163. There is an extra dot at the end of the sentence.

Figures: Please indicate the time duration of the treatments in the figure legends.

Fig. 4. Please change the order of POD and CAT, because CAT is the main enzyme in H₂O₂ decomposition.

Fig. 5. In the future, I would like to suggest the Evans blue staining for the detection of cell death.

Fig. 7. Does chlorophyll content mean chlorophyll a+b?

Fig. 8. Detection of the activity of CAT is missing.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

General comments:

The study was undertaken to improve the knowledge about the use of alginate-derived oligosaccharides to alleviate the damages of simulated acid rain and high temperature during the early vegetative growth of rice plants.

Rice is one of the most widely consumed cereal crops and its cultivation requires a lot of water. Therefore, to ensure food security in a changing climate, we really must find rice varieties that tolerate high temperatures and acid rainwater.  

I am not an English native speaker, but I advise authors to have their paper reviewed by a colleague (or someone) for whom English is the first language because some parts of the manuscript were not easy to understand. Example: page 2, line 49 “…narrower stomachs after exposure of acid rain lower than pH 3…”, please see more details in the specific comments). Nevertheless, the introduction is mostly fine and including the available literature associated with the topic of the study. The experiment was well designed, describing the methods used with enough detail (but other important measurements could have been performed like the gas exchange measurements and chlorophyll fluorescence, for example).

In my opinion, the manuscript presents some interesting findings that fall within the scope of Agronomy. Therefore, I suggest the manuscript for publication after major revision since there are some few points that require improvement or clarification.

Specific comments:

Abstract

Lines 15-17 “Microscopic analysis showed that pH-2 AR injured epidermal of leaves, and more injured structures occurred in leaves under HT condition.”. I suggest to rewrite the sentence to clarify the text (suggestion: “Microscopic analysis showed that pH-2 AR and HT injured leaf epidermis, particularly the bulliform cells, the veins and interveinal regions.”).

Lines 16 and 20 Authors indicate the pH treatments in different ways (PH-2 and PH7). It should be uniform along the manuscript.

Introduction:

Page 2, lines 48-49 “…CO₂ accumulates in intercellular space due to narrower stomachs after exposure of acid rain lower than pH 3…”.  I think that authors wanted to say stomata or stomas instead of stomachs.

Page 2, lines 60- 61 Replace “… and interferes photosynthesis” by “… and interferes in photosynthesis”.

Materials and methods:

Line 93 I Why the rice variety Tianyou 94 998 was selected for this study? Is a high-yielding hybrid rice variety? A locally adapted variety? Previous studies indicate tolerance to stress conditions?

Lines 101, 103 and 105 Replace “green house” by “greenhouse”.

Line 125 Indicate the origin of the AOS solution used in this study. It was produced by the authors in the laboratory?

Results:

Line 238-239 “…low pH rain and high temperature extremely significantly decreased chlorophyll a content” might be replaced by “… low pH rain and high temperature caused a significant decrease (P-value = …) of chlorophyll a content”?

Line 303.  Rice plant is a grass and leaves have a parallel venation arrangement. I think the term “accessory veins” is not the most correct because in parallel venation arrangement, leaves have main veins and connecting between the main veins are the commissural veins (perpendicular to the major parallel veins). Please check this information.

Discussion:

Lines 486-487

The following sentence should be rewritten to improve the clarity of the text “As results of chloroplast being damage and consequent chlorophyll contents decline, the growth and development rice were seriously retarded”.

Line 507

This sentence should also be rewritten: “It’s worthy that there were interaction among rain pH, temperature and AOS (Figure 7), which means that these results were not the simple effect superimposition of these three factors”.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Dear authors,

I think Your  manuscript  is interesting and well written, despite several minor mistakes (please, find highlighted in the pdf attached). I also would like to pay attention to several issues (also highlighted in the text):

• Fig. 9.: what are the difference between "without AOS" in this Fig,. and "high temperature" in Fig 5. Based on the materials and methods, the conditions should be the same, but the the leaves "without AOS" seems to be much better in Fig 9 than "high temperature" in Fig 5.
• Line 365. The  note about AOS effect on SOD when pH<3 seems to be misleading, since AOS treatment increased SOD activity, when rain pH was 2. I see the effect of decreasing pH as stimulating activity of SOD until the specific threshold and inhibiting after crossing it (like it is written in the discussion). Whereas AOS seems to be  a certain factor that enables SOD to work with equal activity regardless of environmental stressor (pH). Maybe it is worth to add a short explanation about that in the discussion. Actually it is written that AOS enhance the activity of antioxidative enzymes, but from these results the effect on SOD seems to be more upregulating and stabilizing than just stimulating.
• It would be interesting to see the AOS effect on CAT activity, It was investigated in the case of pH-temperature treatment (Fig 4). Was it analyzed in the case of AOS treatment?

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors thoroughly revised their manuscript based on my comments. Thank you. 

Reviewer 2 Report

I carefully read the revised manuscript and the authors' responses to my comments. I am satisfied with the improvements that have been made and I think the revised manuscript is ready for publication.