Roles of Exogenous 5-Aminolevulinic Acid and Dihydroporphyrin Iron in Chlorophyll Precursor Synthesis and Chlorophyll-Related Enzyme Activities in Wheat Under Different Light Intensities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

I reviewed the manuscript and the following issues should be noted:

Abstract Part:

1. One or two sentences should be added at the end of the Abstract part as a conclusion.

2. The sentences "In this study, the optimal concentrations for foliar-spray application of 5-ALA and DHFe to promote wheat seedling growth and leaf chlorophyll content were assessed. The optimal concentrations of 5-ALA or DHFe (50 mg/L and 0.01 mg/L, respectively) were applied as foliar sprays to seedlings of wheat ‘Bainong 4199’ (high light efficient) and ‘Zhoumai No. 18’ (average light efficient) under different light intensities." should be revised and also these sentences should be combined  for giving the new important results.

Introduction Part:

1. One or two paragraphs should be given about the effects of different light intensities on plants, especially wheat.

Materials and Methods Part:

1. The source of the plant material should be stated.?

2. Why were the applications applied at the third leaf fully expanded? Give reference or reason?

3. Please, check these sentences "When the third leaf had fully expanded, the seedlings were sprayed with 5-ALA (5, 50, 75, 100, or 125 mg/L) or DHFe (0.001, 0.005, 0.01, 0.05, or 0.1 mg/L) supplemented with Tween 20 (0.01%) as a surfactant" 

4. The authors should define the FWB and FWA formula identification.

5. Where is the calculation formula for Porphobilinogen? It should be given.

6. Please correct the "4°C)" as "4°C.

7. Which was the trial design used in the study? It should be explained in the Statistical analysis subsection.

Results Part:

1. The subsections 3.2, 3.3 and 3.6 should be rewritten by giving detailed results.

2.The figures in figure 2 should be combined if possible.

Discussion Part:

1. The discussion part should be divided into subsections based on the subsections of the Results part.

2. Lines 441-453, 477-487 and 507-514: Font size is different.

References Part:

1. The name of the Triticum aestivum should be written as italic.

Best regards,

Author Response

Comments 1:

 Abstract Part:

1. One or two sentences should be added at the end of the Abstract part as a conclusion.

Response:Thank you for pointing this out. We agree with this comment. Therefore,I have added conclusions to the summary. Add content as follows:”5-ALA promoted the synthesis of chlorophyll precursors by regulating the activities of 5-amino-ketovalerate dehydratase and uroporphyrinogen III synthase. DHFe decreased chlorophyllase activity, thereby slowing chlorophyll degradation and increasing the chlorophyll content”.

 

2. The sentences "In this study, the optimal concentrations for foliar-spray application of 5-ALA and DHFe to promote wheat seedling growth and leaf chlorophyll content were assessed. The optimal concentrations of 5-ALA or DHFe (50 mg/L and 0.01 mg/L, respectively) were applied as foliar sprays to seedlings of wheat ‘Bainong 4199’ (high light efficient) and ‘Zhoumai No. 18’ (average light efficient) under different light intensities." should be revised and also these sentences should be combined  for giving the new important results.

Response: Thank you for pointing this out. But we disagree with this comment. Our experiment consisted of two parts: one was to determine the optimal concentration of 5-ALA and DFHe, and then to evaluate the chlorophyll synthesis using two cultivars with different light efficiency.

Introduction Part:

1. One or two paragraphs should be given about the effects of different light intensities on plants, especially wheat.

Response: Thank you for pointing this out. Light intensity For wheat growth, too weak or too strong light will affect wheat growth. Too weak light can reduce the efficiency of wheat photosynthesis, making the plants grow short and the leaves turn yellow. Excessive light exposure can cause excessive transpiration and rapid water loss from the leaves of wheat plants, thus affecting the growth of wheat. Generally, wheat leaves with high light efficiency are darker in color and contain higher chlorophyll. However, this study was focused on evaluating the effects of 5-ALA and DHFe on chlorophyll synthesis under two different light intensities. The effect of light intensity on wheat is various. This paper only discusses the effect of light intensity on chlorophyll synthesis. Therefore, the author thinks it is not appropriate to add a section to introduce the effect of light intensity on varieties in the introduction.

Materials and Methods Part:

1. The source of the plant material should be stated.?

Response: Thank you for pointing this out. we agree with this comment.The source of the experimental material has been added.

2. Why were the applications applied at the third leaf fully expanded? Give reference or reason?

Response: Thank you for pointing this out. This is an important question. Wheat trifoliate stage is the weaning stage, at this time, the nutrients in the seeds have been exhausted, and spraying 5-ALA and DHFe at trifoliate stage can avoid the influence of the nutrients in the seeds on the experimental results.

3. Please, check these sentences "When the third leaf had fully expanded, the seedlings were sprayed with 5-ALA (5, 50, 75, 100, or 125 mg/L) or DHFe (0.001, 0.005, 0.01, 0.05, or 0.1 mg/L) supplemented with Tween 20 (0.01%) as a surfactant" 

Response:Thank you for pointing this out. We have checked and corrected it.

4. The authors should define the FWB and FWA formula identification.

Response: Thank you for pointing this out. We have checked carefully. When the calculation formula is given in the article, We have subsequently given an explanation.

5. Where is the calculation formula for Porphobilinogen? It should be given.

Response:Thank you for pointing this out.The formula has been supplemented.

6. Please correct the "4°C)" as "4°C.

Response: Thank you for pointing this out. We have corrected.

7. Which was the trial design used in the study? It should be explained in the Statistical analysis subsection.

Response: Thank you for pointing this out. The experimental design has been supplemented

Results Part:

1. The subsections 3.2, 3.3 and 3.6 should be rewritten by giving detailed results.

Response: Thank you for pointing this out. We disagree with this comment. For the 3.2, the experimental results of spraying 5-ALA and DHFe on plant weight gain are mainly analyzed, and the main results of the experiment are given by comparing the treatments in this part. For 3.3, mainly analyzing the results of the effect of spraying 5-ALA and DHFe on chlorophyll a, chlorophyll b and chlorophyll a/b in plants, we compared the data differences between the different treatments and between the treatments and the control. The main results of spraying 5-ALA and DHFe with combined treatments are presented. For 3.6, the main analysis was the correlation between the parameters measured after spraying 5-ALA and DHFe, aiming at the correlation between the parameters after spraying treatment. The results have shown the relationship between 5-ALA and DHFe and the parameters. The data analysis has served its purpose.

2. The figures in figure 2 should be combined if possible.

Response:Thank you for pointing this out. We disagree with this comment. For Fig. 2 involving the enzyme activity changes of each of the three enzymes under two light intensities, the single analysis is more concise.

Discussion Part:

1. The discussion part should be divided into subsections based on the subsections of the Results part.

Response: Thank you for pointing this out. During the discussion in this paper, the content has been analyzed. The first paragraph analyzes the spraying concentration of 5-ALA, the second paragraph analyzes the spraying concentration of DHFe, the third paragraph analyzes the effect of spraying under two lighting conditions, the fourth to eighth paragraphs analyze the precursors combined with the chlorophyll synthesis step, and the ninth paragraph ends with the analysis of the correlation between the precursors of chlorophyll synthesis. The author believes that the analysis content of this part is coherent, and it is best not to add subheadings.

2. Lines 441-453, 477-487 and 507-514: Font size is different.

Response: Thank you for pointing this out. I have corrected.

References Part:

1. The name of the Triticum aestivum should be written as italic.

Response: Thank you for pointing this out. I have corrected all references for relevant content.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, “Roles of exogenous 5-aminolevulinic acid and 2 dihydroporphyrin iron in chlorophyll precursor synthesis and 3 chlorophyll-related enzyme activities in wheat under 4 different light intensities,” authored by Hu et al., examines the impact of foliar application of 5-ALA and DHFe in wheat. I found the manuscript to be well-written in general and can be accepted for publication. A couple of points that confused me about the study are detailed below. The authors should address these.

1)    The formula for the calculation of RCRB is presented as % in the Methods section. However, Fig. 1A legend does not indicate RCRB as percent. Authors should clarify whether the data presented are percent change or otherwise. The same problem exists in Table 1. There should not be any discrepancies in the method followed and data presented.

2)    Is there a reason for choosing light intensities of 12.5 μmol.m⁻².s⁻¹ and 1000 μmol.m⁻².s⁻¹?

3)    I find the discussion section to be unnecessarily lengthy. Authors can summarize the discussion section.

Author Response

Comments 1:

The manuscript, “Roles of exogenous 5-aminolevulinic acid and 2 dihydroporphyrin iron in chlorophyll precursor synthesis and 3 chlorophyll-related enzyme activities in wheat under 4 different light intensities,” authored by Hu et al., examines the impact of foliar application of 5-ALA and DHFe in wheat. I found the manuscript to be well-written in general and can be accepted for publication. A couple of points that confused me about the study are detailed below. The authors should address these.

•  The formula for the calculation of RCRB is presented as % in the Methods section. However, Fig. 1A legend does not indicate RCRB as percent. Authors should clarify whether the data presented are percent change or otherwise. The same problem exists in Table 1. There should not be any discrepancies in the method followed and data presented.
• Response :Thank you for pointing this out. We agree with this comment. Therefore, we havemade a correction. For the calculated values of RCRB, numerical values are more suitable for the comparison of the significance of differences between treatments in the experiment.So the experiment uses numerical display. The mark (%) by the author in the paper is wrong and has been deleted and corrected.
• Is there a reason for choosing light intensities of 12.5 μmol.m−2.s−1 and 1000 μmol.m−2.s−1?
• Response :Thank you for pointing this out.The fluorescent light intensity of the fluorescent lamp is marked as 1000LX=12.5μmol.m⁻².s⁻¹, 12.5μmol.m⁻².s⁻¹is the weak light quantity. Considering the growing season of wheat, the external light intensity can reach a value of 800~1250μmol.m⁻².s⁻¹.So we chose 12.5μmol.m⁻².s⁻¹ and 1000μmol.m⁻².s⁻¹ as the light intensities for weak and strong light.

3)    I find the discussion section to be unnecessarily lengthy. Authors can summarize the discussion section.

Response :Thank you for pointing this out. The author has tried his best to simplify the discussion

Author Response File: Author Response.pdf