Coronatine Modulated the Generation of Reactive Oxygen Species for Regulating the Water Loss Rate in the Detaching Maize Seedlings

Round 1

Reviewer 1 Report

The manuscript by Yu et al. is devoted to investigation of influence of coronatine on ROS homeostasis, water balance, and antioxidant regulation in plants under drought stress. It is interesting work; however, there are a few remarks:

1. P. 2, lines 79, 84: It is necessary to put dots and spaces. In general, you need to carefully read the text for misprints.
2. P. 3, line 95: Zea mays L.
3. P. 3, line 114: N₂
4. P. 4, line 151-154: A brief description of the technique should be given.
5. P. 5, lines 200, 214: What wavelengths were used?
6. P. 5, line 220: misprint "Stuednt’s"
7. P. 5, line 227 and further in the text: maybe not “after treatment”, but “after start of the treatment”?
8. P. 6, Figure 1 (line 234): not “Time after treatment (h)”, but “The duration of treatment” or something else?
9. P. 6, Figure 2: It is necessary to give the decryption "СK".
10. P. 12, line 327: not “ROS fluorescence intensity”, but “H2DCF-DA fluorescence intensity”
11. I think, a little scheme explaining a possible mechanism of the COR influence is needed in the Discussion section.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript describes the effects of coronatine on maize leaves under drought condition, at the molecular and cellular levels. Please find below my comments and suggestions.

• Figure 2 A and C: the treatment applied should be indicated for each leaf.
• Line 270: “COR significantly increased the expression levels of ZmDREB2A compared to control under drought stress (Figure 4A).” The graph does not show an increase of expression in COR treated leaves.
• Line 273: “Similarly, the expression of ZmcAPX and ZmCAT1 were also induced by 273 drought stress, while COR improved greater the expression levels of ZmcAPX and 274 ZmCAT1 than control (Figure 4B and C).” The graphs do not show an increase of the expression. Please clarify.
• Figures 1, 5 and 8B: Statistics should be shown.
• Figure 5: I find hard to distinguish the lines corresponding to the different conditions. Perhaps colors could be used.
• Line 285: the known function of DMTU should be explained.
• The function of the different genes analyzed should be mentioned before the results.
• Importantly, the concentration of COR used should be mentioned in the legend of all figures.
• Each abbreviation should be explained.
• Abbreviations should be limited in the abstract.
• Line 374 and line 428: “COR could relieve ROS accumulation 374 in plants exposed to PEG treatment.” I do not think that “relieve” is adapted here.
• Line 436: «epixermis » should be « epidermis », I guess.

Author Response

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Reviewer 3 Report

The authors of the paper “Coronatine modulated the generation of reactive oxygen species for regulating the water loss rate in the detaching maize seedlings” performed a study on the effect of coronatine (COR) on the drought tolerance in maize. They showed that the effects of this phytotoxin depend on its concentration: high ones increased water loss while low ones reduced it. A 0.001 µM concentration of COR has a priming effect on the plant reducing the oxidative stress caused by drought. In a particular way, it modulates the transcriptional profile upregulating specific genes involved in the drought tolerance. Even the enzymatic activities involved in the detoxification of reactive oxygen species (ROS) are increased by COR during the stress. Interestingly, the author performed a preliminary analysis that suggests a possible role of COR in the ROS metabolism in the guard cells.  The author's results can improve the knowledge of the molecular mechanism of COR in drought stress. But this article must be reconsidered despite all results that the authors obtained. The material and methods are not described clearly. The results are not well showed and it is quite confusing all the presentation All the text needs to be rewritten, improving the English but overall, the scientific soundness. The authors are invited to resubmit the paper after a deep revision.

Here some questions and hints for the authors:

1. It would be better to improve the abstract. It is not clear for the reader as all the text.
2. Write the full name of the antioxidants DPI and DMTU in the abstract, please.
3. Write the full name of ROS at line 15, please.
4. Why did you study DPI and DMTU? You might just anticipate before the discussion.
5. Typos for H₂O₂ and O^.2-at line 23 of the abstract. The same kind of typos is present in all the text.
6. You should write the full name of DAB and NBT on line 23.
7. The words abscisic acid and methyl jasmonate are not appropriate as keywords since you did not perform any analysis about them. You only cited the methyl jasmonate in the discussion.
8. You should add references for the sentence at lines 36-37 and 44-45.
9. Typos at line 57.
10. Report previous studies on the drought priming in maize in the introduction.
11. Typos at line 58.
12. You should use the italics font for the scientific name of plants and bacteria. Check all the text and bibliography too, please.
13. Typos at line 69.
14. The word "documents" at line 70 is not appropriate.
15. Typos at line 79.
16. Why is still faint the role of the COR in the modulation of the ROS production?
17. How does the COR modulate the antioxidant regulation? You explained it in the discussion, but it would better to explain in the introduction.
18. Typos at line 84.
19. References at line 85.
20. Typos at line 95, “Zea mays” instead of “Zea may”.
21. At line 100, control the sentence.
22. Can you report a reference (line 102) that states the treatment of the detached leaves in water for 1 hour can eliminate the wound stress?
23. “Weighed” instead of “weigh” at line 105.
24. Which inhibitors do you mean at line 107?
25. Did you use the first leave for the water loss analysis while the second leaves for qRT-PCR and enzymes activities etc?
26. It is not well described how you treated the protoplast.
27. N₂ instead of N2 at line 114.
28. There is a typo at line 117.
29. Re-check the paragraph “Plant growth and treatments”. It was not written clearly.
30. It would be better to use the word isolation instead of preparation at line 119.
31. Could you indicate the unigene IDs for your genes, please?
32. You might indicate the sequences of the primer in a supplementary table.
33. Why did you choose β-tubulin? Is it known to be stable during drought stress?
34. How did you design the primer pairs?
35. Which is the primer concentration that you used?
36. Which is the volume of cDNA that you tested?
37. Which is the efficiency of the primer pairs?
38. How did you calculate the fold increase of the gene expression?
39. How many biological replicates did you test?
40. How many technical replicates did you use?
41. Which are your calibrator samples?
42. Improve the paragraph of the “RNA preparation and real-time quantitative PCR analysis”. Use the term isolation instead of preparation.
43. Use the italics font for “in vivo”
44. What did you mean for A416 at line 151?
45. Typo at line 152
46. How did you make the calibrator curve for the Bradford method?
47. Which was the concentration of your proteinic extract? How many µg of total protein did you use for the enzyme assays?
48. Typo at line 158.
49. You did not perform the PEG treatment for stomata aperture measurement, did you?
50. Recall that the plants were etiolated as described previously at line 206.
51. Write the full name of BSA, please.
52. Write the full name of H2DCF-DA, please.
53. The material and methods are not described clearly. Re-check all this section.
54. What did you mean for three-leaf seedlings at line 224?
55. The control plants for the experiment of the water loss are detached leaves treated only with PEG, aren’t they?
56. Did you test the water loss of only detached leaves (not treated with COR and PEG)? Did you test the water loss of the CK and COR detached leaves (as you named in the next section of results)? Maybe, you could show the results in the supplementary data.
57. Are all the experimental points of the 0.001 µM COR statistically different?
58. There is a typo in the description of the y-axis, “of” instead of “0f”.
59. It might be better to specify “PEG treatment” on the x-axis.
60. You might simplify the legend of figure 1.
61. Why did you choose the 0.001 µM COR for the next experiment instead of 0.01 µM?
62. I would have expected that the water loss was lower in 0.01 µM than in 0.001 µ Why did this happen?
63. You should improve the quality of Figures 2 A and 2 C. You should design a legend of the treatment and specify that the first four leaves on the right are the first detached leaf. The same is for the second four leaves that they are the second leaf. The improved figure you might display in the supplementary section. By now, the figure is not clear to the reader.
64. You need to describe better this experiment, especially the treatments, in the material section.
65. What do you mean for normal conditions at line 244?
66. Did you mean figure 2C instead of figure 2B at line 245?
67. Re-check the order into the panel of figure 2 and its correspondence in the text, please.
68. Did you mean figure 2B instead of figure 2D, didn’t you?
69. Improve the legend of figure 2. You should indicate what do you mean for CK, COR, PEG, COR + PEG. There is no need to report the material and methods in this legend.
70. You stated that there is a slightly different level of CAT activity between CK and COR detached leaves, but this difference is not statistically significant.
71. What do you mean for control plants under drought stress in line 265?
72. It seems to me that you didn’t describe the double treatment detached leaves (COR+PEG), doesn’t it?
73. Specify the treatment in the legend of figure 3. What do you mean for CK? Which is the difference with PEG?
74. Why did you decide to test these genes? They are known to be involved in some specific pathway for the tolerance to the drought in maize?
75. How many DREB genes are present in maize?
76. Why did you test this isoform of ZmDREB?
77. Which enzymes are codified by ZmCAPX and ZmCAT1?
78. What did you mean for normal conditions at lines 272-273?
79. The relative expression of the genes ZmcAPX and ZmCAT1 for the COR treatment are not statistically different from the CK treatment except for ZmcAPX at 1 h.
80. There is a typo in the legend of the figure PEG+COR instead of PEG+CPR.
81. It is not clear the legend of figure 4. What do you mean for the relative expression of the control plants versus the control plants grown under normal conditions?
82. Improve the quality of figure 5. Could you merge the two figures 5A and 5B? Why are the results for CK and COR quite different in Figures 5A and 5B?
83. Can you describe why DMTU and DBI are expected to contrast the COR effects as you made in the discussion?
84. Which genes are the ZmRBHOB? For what enzymes do they codify? In which step of the NADPH oxidase-dependent production are involved?
85. Why aren’t there the PEG and PEG +COR treatments in this qRT-PCR analysis?
86. Why the data are different for the panel A and B for CK and COR?
87. It’s not clear to me what you mean for CK in figure 8C.
88. Control the figure 8B. The shape of the indicator is square at 10 and 12 min for the CK treatment instead of being round.
89. The standard deviation is not indicated for all the data in panel 8B.
90. What do you mean for exposed to the growth camber on line 355?
91. What do you mean for water holding capacity?
92. You can merge the section of the conclusions with the lines from 423 to 431.
93. Re-write the discussion part from lines 407 to 431.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript has been greatly improved. I do not have further suggestions.

Author Response

Thank you for the careful and critical suggestions. 

Reviewer 3 Report

Dear editor, the author of the paper “Coronatine modulated the generation of reactive oxygen species for regulating the water loss rate in the detaching maize seed” answered all my questions. They were very ready to modify the text. It should be resubmitted to have the quality standard of your journal. The text in the material and methods are still not well described. The methods of the treatments of detached plants, of the protoplast, have to be implemented as well as the methods of the real-time analysis. The results are still not clearly presented using not an appropriate scientific language. For example, great confusion is generated using the term control both for well-watered and drought seedlings for the biochemical results as well as for the real-time analysis. Furthermore, the experimental design has some problems, for example, lack of the study of the rate of water loss in the detached plants only treated with different concentrations of COR or with the inhibitors DPI and DMTU; absence of the ZmRBOH genes expression during drought stress only and in combination with COR; no analysis of stomatal closure during drought in leaves and protoplasts, no study of the singular effect of DPI and DMTU for leaves. Despite this, the authors performed extensive research work and showed good preliminary data. Overall, the results of this paper can contribute to deciphering the role of the coranatine in the ROS signaling during drought. So, the authors are invited to re-edit the text.

Here some suggestions and tips for the authors.

1. You could add the word drought as keywords.
2. Re-check the grammar of lane 80. “Acts in” instead of “acts a”.
3. You might remove under drought stress at line 84. It is a repetition since you just writing about drought tolerance.
4. At line 143 treatments instead of treatment. You might change the sentence at line 143 in “after having identified the best COR concentration,..”.
5. You might change this sentence “to detect the ROS accumulation” in “histochemical detection of H₂O₂ and O₂^.-“.
6. Change “stetms” in ”stems” at line 150.
7. A typo at line 157, correct “every one hour” in “every hour”.
8. Indicate in paragraph 2.2 the volume of cDNA, the final concentration of the primers. Their efficiency. The thermal cycle that you used. The number of technical and biological replicates. The reference of the previous studies using the gene β -tubulin as a reference gene during drought stress in maize. This is very important information to validate your data.
9. Add COR after 0.001 µM at line 180.
10. Remove the word “physiological” from the title of paragraph 2.4 because you described the physiological analysis in the next paragraph.
11. Specify the control treatment as detached leaves treated with water. See number 16 too.
12. Specify the absorbance at 415 nm at line 255 instead of the abbreviation A415.
13. Typo at line 318 “keptp”.
14. In line 353, the verb “selected” is not appropriate. It should be better to use “analyzed”.
15. In line 355, it might be better to replace the word treatment with drought or water stress. It is useful for the reader that you mean PEG treatment. I realized that you mean that as expected there was a water loss during the drought in all your samples even in presence of COR. Nevertheless, the sentence at lines 354-355 is not quite right. You stated that the rate of water loss increased regardless of COR-treated seedlings. But after your results showed that this parameter increased or decreased in a COR dose-dependent manner.
16. At lines 359-360, maybe you might not use the adjective “control” but “not-treated with COR”. This might create confusion since in the next experiments you are going to indicate as control the detached plants immersed in only water for the time of treatment. Here the right controls would have been the detached seedling immersed in only water and with different COR concentrations in parallel. In this way, you would have clearly shown that the water loss is only due to the PEG and COR can only modulate it. But I see by your answers that you aimed to determine the best COR concentration that decreased the rate of water loss.
17. Typo at line 385. Capital letter for “student’s”.
18. You might write something like “well-watered conditions” and, ”drought treatment” instead of “normal conditions” and ”PEG treatments” at lines 395 and 398, respectively. It should be better for the reader. This is for all the text.
19. For the biochemical analysis, it might be confusing the description of the PEG leaves as control too since you just represented the well-watered plants not treated with COR (CK) as control. For a reader is instinctive to think about only one category of control rather than two categories for the experiment shown in figures 2B and 2D. You might describe CK as well-watered plants, COR as well-watered plants treated with COR, PEG as water-stressed plants, PEG+COR water-stressed in presence of COR
20. In line 397, typos “contents” instead of “content”.
21. You might remove the sentence “as described in the Materials and Methods section” at line 419.
22. You might indicate in the figure that you are making a comparison between well-watered, and water-stressed leaves, drawing the first line under the two first histograms and the second one under the reaming histograms.
23. In line 416, “Coronatin (COR) treatment induced H₂O₂ and O₂ ^-·accumulation in leaves of maize plants with PEG treatment” this partially right since even drought-induced this accumulation. You might describe “H₂O₂ and O₂ -accumulation in leaves of maize plants after treatments with COR in well-watered and drought plants”.
24. In line 418, you might specify that the “second” leaves were homogenized.
25. It might be better to use the adjective “well-watered” instead of “normal” in line 426.
26. The same above considerations for the caption of figure 3.
27. Eliminate “is” at line 438.
28.  In line 438, you might highlight that ZmDREB2A regulates the drought-responsive genes besides to be upregulated reporting the references.
29. Eliminate “compared to the control” at line 442. “COR significantly increased the expression levels of ZmDREB2A under drought stress conditions”.
30. Change “to the control” to “the only drought-stressed plants”.
31. The same above considerations for the caption of figure 4. It would be better to use the adjective well-watered instead of normal. It gives more emphasis to your objectives. It should be useful for the reader not to describe the PEG-treated plants as a control.
32. In the caption of figure 4, you have to indicate your biological calibrator better. As you answered in the previous response, the calibrators are the well-watered detached plants at 1h. Instead, you described as you set RQ of CK at 3 and 5 h equal to 1 too. You might better describe this in the materials and methods section.
33. At lines 473-482, you are right that the controls for the rate of water loss experiment (in presence of DPI and DMTU) are the drought-stressed plants, but it creates confusion to the reader. You might indicate CK with WS (Water-stressed) for continuity with the text. I’ll repeat even if it quite obvious that the well-watered wouldn’t have last water, you should have estimated it as control.
34. For figure 5, you might change the description of the x-axis in PEG treatment as in figure 1.
35. There is a contradiction between the description of the treatments in the text (lines 473-482) of the results for figure 4 and that in the corresponding materials and methods: “To study the effects of the inhibitors diphenyleneiodonium chloride (DPI) and dimethylthiourea (DMTU), the detached plants were pre-treated with 0.001 μM COR for 12 hours, then treated with 2.5 mM 154 (DMTU), or 2.5 μM (DPI) for 8 h, respectively, and then subjected to 10% (w/v) polyethylene glycol (PEG 6000) solution. To calculate the water loss rate, the detached plants were weighted every one hour for 8 h”. You should add that the second group of plants wasn’t pretreated with COR.
36. You should describe the symbols CK, DPI, etc in the legend of figure 4.
37. You might change the title of the paragraph. “COR induced NADPH oxidase of the ROS signaling gene network….”. I don’t know if the term “production is appropriate”.
38. You might describe briefly the ZmrBOHS genes as you made in the previous answers “ZmRBOHs genes encoding NADPH oxidases in maize plants which catalyze the production of superoxide”. Stress that this gene family is involved in the ROS signaling network reporting the reference,
39. At lines 510-512, “The expression levels of ZmRBOHA and ZmRBOHB were slightly induced by COR at 3 to 12 h after treatments”. This sentence is not quite correct. There is no significance for ZmRBOHA at 3 and 12 h. For ZmRBOHB, only at 3 h.
40. For all the real-time figures, you have to change the name of the x-axis or in “Time of treatments (h)” or simply in “Time (hours)”. Because the plants are subjected to different types of treatments.
41. In line 519, there is a mistake “to ZmActin transcript levels”, your endogenous gene is β-tubulin.
42. Improve the legend of figure 6. The relative expression of CK is equal to 1, not the sample.
43. You should change the order of the results of paragraph 3.3. At the beginning the qRT-PCR analysis, then the rate of water loss, and in the end the stomata closure.
44. At lines 522-523, you should highlight the goal of your analysis to observe whether COR and ROS are related to the regulation of stomatal action inhibiting the activation of NADPH oxidase in well-watered plants.
45. In lines 522-529, there is no correspondence with the description of the treatments in the corresponding material and methods section. “To study the effects of COR on water loss, the detached plants were pre-treated with 0.001 μM COR for 12 h and then treated with 2.5 mM dimethylthiourea (DMTU), 2.5 μM diphenyleneiodonium chloride (DPI) for 8 h. Detached plants were treated with distilled water under the same conditions for the whole period and served as controls for the above”. You have to describe how you treated the detached plants for only DPI/DMTU treatments in absence of COR priming for 12 h.
46. At lines 528-529, this sentence “However, DPI associated with COR increased the stomatal apertures by 1.3% compared 528 to the control” is not quite right since there is no significant difference.
47. Remove at line 533 “the same time point”. Also, in the caption of figures 2, 3, and 8.
48. Indicate a reference at line 536, “Since stomatal closure is accompanied by increased ROS level in the guard cell”.
49. Re-modulate this sentence “relative to the control in the DPI-incubated protoplasts, and by 19.2% compared to control in the DMTU- incubated protoplasts”. Maybe you could write “relative to the only DPI-incubated protoplasts”….in the same way for DMTU.
50. You have to modify figure 8C. Use a different color for the histograms: white for DMTU+COR, grey DPI+COR. Use a different layout of the histogram for figure 8C and. Don’t show in pair but 6 different histograms. In this figure, we showed four different categories of CK: 1^st the detached leaves well-watered, 2^nd the detached leaves with COR, 3^rd those with DPI, 4^th with DMTU. You might show the protoplast without COR priming treatment and drawing a line under the three boxes and the second three histograms with COR treatment drawing a line under the remaining histograms. Or not indicate the black boxes as CK but as not-COR treated.
51. You might specify in the y-axis of figures 8A and 8C “relative fluorescence intensity (%) in leaves” and “in protoplast”, respectively.
52. For figure 8B, you could write only time for the x-axis.
53. Eliminate “using ROS probe H2DCF-DA” at line 823.
54. ROS production which “are” induced by COR at line 825.

Author Response

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Author Response File: Author Response.docx