Coronatine Enhances Stalk Bending Resistance of Maize, Thickens the Cell Wall and decreases the Area of the Vascular Bundles

Round 1

Reviewer 1 Report

In this study, the authors study the effect of the phytotoxin, coronatine, on maize stalk bending, and lignin related traits.  They report that the coronatine increases stalk pushing resistance, and decreases plant and ear height as the concentrations of coronatine increases in two inbreds in field grown studies.  Cross sections of B73 internodes showed increased lignin content, and under scanning electron microscopy, some developmental differences between B73 internode cross sections treated with coronatine versus corresponding control over time.  I really enjoyed reading this manuscript and feel it offers good insight into the effects of coronatine on maize.

A few comments include:

1. The title might should be revised, since stalk lodging resistance was taken in the field with different inbreds than the B73 that was used for the microscopy sections/analysis.  While it is logical to assume that one begets the other, different environments and different inbreds were used.  And there is no proof that one caused the other.  But that all three conditions occurred, 2 in different conditions than the third. Suggestion includes 'Coronatine enhances stalk bending resistance, cell wall thickness and vascular bundle area'
2. There are typos throughout the manuscript that can be fixed pretty easily. Have someone go through it and hit them all. While the majority of the writing is quite good, there are these little typos here and there, spacing issues between words, before commas, and eg.
   1. page 2 line 42, risk not rist
   2. Line 51 previous research has, not researches have..
   3. Line 55 synthesis not ynthesis,
   4. Page 3, line 119, was not were

3. Results section, please indicate in the results whether you are talking of the field or greenhouse study
4. Figure 2, Plant height and ear height, can you put the two years together? Why keep them separate?  Also include if the bars are standard error bars. And be sure the letters are positioned on top of the bars.
5. Table 1. Why keep the years separate?
6. Figure 3. Are these standard error bars?
7. Figure 4. Would you expect something different with a different concentration of COR?  Why did you use COR10?
8. How related are the 2 inbreds in the field to B73? Are they all temperate?
9. Figure 7. Need to include standard error bars on the data points?  Is this a significant difference?

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The study presented by Li et al. analyses the effects of coronatine on lodging resistance. Next to morphological criteria, they analyzed stalk strength, hormone contents, lignin content and cell wall morphology. While the study is potentially interesting, data analysis and data presentation are rather poor and the manuscript lacks quantitative data to support the conclusions. Please find my specific comments in the following:

• Introduction
  • Please specify the conditions under which coronatine acts as phytotoxin vs a plant growth regulator
  • I’m not quite sure about the reasoning in choosing coronatine? The introduction states that plant growth regulators can influence lodging stress in various crops (please state which ones with proper citations). Was that the only reason to choose coronatine, because it can have effects as plant growth regulator? Please explain better.
• Material/Methods:
  • Why were different maize inbreds used in field trials vs greenhouse experiment?
  • Why was Tween used in the greenhouse, but not in the field to spray coronatine?
  • Why was the third basal internode used?
• Results
  • Please provide images of the plants after coronatine treatment
  • Why were there such big differences in plant height reductions between the experimental years (from Cor30 to Cor60 there was a further reduction in 2019, while there was an increase in 2018 for example), or dry weight of the basal internode? It appears to me that coronatine treatment is very variable, which hinders conclusion from only 2 replications.
  • Why are the 2 years not combined?
  • Please define rind puncture strength? Is that supposed to mean ring penetration strength? Also, the rind puncture strength/ring penetration strength ranking as outlined in the text, does only correspond to XY335 in 2019, but not to any other given data in table 1. Please correct
  • Table 1: no standard deviations or errors are given. I also don’t understand the significance as given in table1 regarding source of variation
  • Figure4: I think it is really hard to conclude stronger fluorescence qualitatively. Maybe some sort of fluorescence quantification can help. Please also label the images better (where is the xylem and the schlerenchyma in the presented images). Also how many plants showed that distribution?
  • Figure5: How do you conclude quantitative measures without measuring? For example how do you know that the area of the vascular bundle decreased in cor treatment, or that this is due to VBS thickening. Also how do you see that the cell walls were thinner and that there were many holes in the cell wall. I cannot draw the same conclusions from the presented images. Please provide quantitative measurements. Also please explain differences in the control.
  • Figure6: label with A-D. Like for figure 5: Please provide some quantitative measures.
  • What biological significance has a higher angle in the pressure stress experiment?
  • Figure7: please provide standard deviations/errors. Is the increase in the curve statistically significant? Also, why are the cor sample points at slightly different angles (x-values) than the control?
• Discussion
  • Line 356: COR reduced the diameter of PX and the are of vascular bundle: the presented data does not support this
  • Line 379: COR influences lignin accumulation and cell wall biosynthesis, which led to inhibition of internode elongation: This is not supported by the data: lignin accumulation if at all happened very early on and if correct appears only at a very specific timepoint. No direct test, whether lignin accumulation leads to inhibition of internode elongation was performed
  • In general, the discussion is very speculative; unfortunately, some very cool results are not discussed (for example the hormone results)

Author Response

please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I would like to thank the authors for replying to all of my comments. Unfortunately, many of my comments were only answered in their cover letter, yet not changed in the manuscript, which still hinders proper interpretation of the presented results in the manuscript. Additionally, the presented data in my eyes does still not comply to scientific soundness (missing standard errors and proper statistics in multiple experiments). While the study is potentially interesting, with the improper presentation of the results I do not suggest publishing in the current form. In addition it appears that none of the figures were changed from the previous version, although stated in the response to my review. My detailed comments are listed below:

1) As stated by the authors: COR can induce leaf chlorosis, increased ethylene generation, and accelerated senescence, while low concentrations can have growth promoting effects. My initial question about the specific conditions or concentrations of COR that are needed for either inducing leaf chlorosis or having growth promoting effects was not answered, yet are instrumental in understanding the different concentrations used in the study. How do concentrations of 10, 30, and 60uM affect plant growth or differently said are they inducing leaf chlorosis = acting as toxin or are they promoting growth? For example in your comment on including plant pictures you mention that 30uM already was poisonous to plant growth. This is important information that should be included in the manuscript.

2) Different inbred lines were used in the study. While my initial question, why these inbred lines were used, is answered in the response to the reviewers, these explanations were not added to the manuscript. In my eyes it is an important information that one of the inbreds is lodging susceptible and the other one is lodging resistant and is a pivotal information for the manuscript and the interpretation of the data.

3) Like comment 2) the reasoning of using the third basal internode is given in the response to the reviewers letter, yet not added to the manuscript.

4) The authors state that they have included a plant picture of COR treatment in Fig. 2, which does not hold true for the revised version I obtained.

5) A method description of how to obtain rind puncture strength or ring penetration strength is not given in the material and methods section, but again in the response to the reviewers letter. In case the terminology for those are given differently in the material/method section it should be adjusted and kept uniform to not confuse readers.

6) Table 1: In my eyes proper scientific practice is to include standard deviations or errors in statistical data. My original comment was neglected and it was stated that the missing of standard deviations and errors is due to the table being large. I don’t think that this is a valid justification for not presenting these statistical measures, especially if you can easily provide them.

7) Fig. 4: I am a little confused. The authors state in their response, that they have changed Fig. 4 by labelling the appropriate cell structures. I cannot see any changes in Fig. 4. The authors also state that they have included a table 2 with quantitative measurements of vascular bundle and protoxylem vessel area. While I see this table in the response letter it is not included in the revised manuscript version that I have obtained. Even if it was, table 2 in the response letter simply lists averages, that again do not provide standard deviations or errors, which is not good scientific practice.

8) Fig. 6 was said to have been labelled, which is not correct.

9) Fig. 7: My comment about statistical significance or introducing standard deviations was unfortunately ignored.

Author Response

• As stated by the authors: COR can induce leaf chlorosis, increased ethylene generation, and accelerated senescence, while low concentrations can have growth promoting effects. My initial question about the specific conditions or concentrations of COR that are needed for either inducing leaf chlorosis or having growth promoting effects was not answered, yet are instrumental in understanding the different concentrations used in the study. How do concentrations of 10, 30, and 60uM affect plant growth or differently said are they inducing leaf chlorosis = acting as toxin or are they promoting growth? For example in your comment on including plant pictures you mention that 30uM already was poisonous to plant growth. This is important information that should be included in the manuscript.

Response: Line51 “ approximately 10000 folds more MeJA” was added  

 Line 53-54” Spraying 0.1 umol L^-1 COR on the leaf could enhance drought tolerance in wheat by maintaining high photosynthetic performance [21].”was added

Line 59-61” 20 umoL^-1 COR induced three-fold higher amounts of anthocyanin and 2 umoL-1 inhibited root growth in tomato seedlings [30]” was added

Line 62-64 ”There was a strong induction of the alkaloids in a concentration-dependent manner during COR treatment with various concentrations of COR (0.1-100 umol L-1) and 100 umol L-1 COR increased nicotine concentration of dry mass in tobacco [26]” was added

In Results : Line 217 “However, when COR concentration was greater than or equal to 30 umol L-1 (≧30 umol L^-1) showed the growth of maize plants was poisoned, over-dwarfed the plants, weak stem, reduced biological yield and resulting in a smaller panicle or even no panicle of maize plants (Figure 2). The results indicating that COR had a concentration-dependent manner in effect on maize plant during COR treatment with various concentrations.” was added.

• Different inbred lines were used in the study. While my initial question, why these inbred lines were used, is answered in the response to the reviewers, these explanations were not added to the manuscript. In my eyes it is an important information that one of the inbreds is lodging susceptible and the other one is lodging resistant and is a pivotal information for the manuscript and the interpretation of the data.

Response: Line 108-109 the statements of  “ZD958 and XY335 were used as experimental materials.”g were corrected as “Hybrid maize cultivars"XY335"(lodging-susceptible) and"ZD958" (lodging-resistant) which are widely planted in China, were used as experimental materials to research the effect of COR affects on agronomic traits and that related lodging resistance of maize.”

Line117 ”its gene sequence has been known” was added.

• Like comment 2) the reasoning of using the third basal internode is given in the response to the reviewers letter, yet not added to the manuscript.

Response: Line 130-134 “We sprayed COR onto maize foliar surfaces at the V7 stage, the seventh internode (the basal 3rd internode on the ground) began to elongate at this time because the internodes below the first four leaves never elongate. And the quality traits of the basal third to fifth internodes exhibited a significantly correlation with lodging resistance of maize, so the basal third internode was used to evaluate the quality of stalks.” was added.

• The authors state that they have included a plant picture of COR treatment in Fig. 2, which does not hold true for the revised version I obtained.

Response: Now We have added the plant image in the Figure2.

• A method description of how to obtain rind puncture strength or ring penetration strength is not given in the material and methods section, but again in the response to the reviewers letter. In case the terminology for those are given differently in the material/method section it should be adjusted and kept uniform to not confuse readers.

Response: Now we have changed and kept it uniform for “rind puncture strength”

Line 138  The statements of “ring penetration strength” have changed to “ rind puncture strength”,“ring penetration strength” in the table2 also have changed to “ rind puncture strength”

We should like to thank you for your helpful comments.

• Table 1: In my eyes proper scientific practice is to include standard deviations or errors in statistical data. My original comment was neglected and it was stated that the missing of standard deviations and errors is due to the table being large. I don’t think that this is a valid justification for not presenting these statistical measures, especially if you can easily provide them.

Response: We have added the standard errors in table1.

• 4: I am a little confused. The authors state in their response, that they have changed Fig. 4 by labelling the appropriate cell structures. I cannot see any changes in Fig. 4. The authors also state that they have included a table 2 with quantitative measurements of vascular bundle and protoxylem vessel area. While I see this table in the response letter it is not included in the revised manuscript version that I have obtained. Even if it was, table 2 in the response letter simply lists averages, that again do not provide standard deviations or errors, which is not good scientific practice.

Response: We have labeled Fig4 and added table2 the quantitative measurements with standard errors of vascular bundle and protoxylem vessel area in Line 306.

8) Fig. 6 was said to have been labelled, which is not correct.

Response: Fig.6 has been labelled.

9) Fig. 7: My comment about statistical significance or introducing standard deviations was 

Response: Data plotting with standard deviations for Fig7.

Special thanks to you for your rigorous academic attitude.