Effect of 5-Aminolevulinic Acid (5-ALA) on Leaf Chlorophyll Fast Fluorescence Characteristics and Mineral Element Content of Buxus megistophylla Grown along Urban Roadsides
Round 1
Reviewer 1 Report
This is an interesting potential application for 5-ALA. Some interesting results are presented. There is much detail missing regarding sampling, and without this it is difficult to assess the results. Editing by a native English speaker is needed.
Line 47: Is there also salt applied to roadways in the winter?
Line 70: It would be helpful to include a 1-2 sentence description of this species.
Line 78: Had these plants been growing in this location for 6 years, or were they planted just prior to the experiment?
Line 81: How many individuals were present in the 100 m long plot?
Lines 83-84: What is meant by “generally surveyed”? Please describe in detail what measurements were made and how.
Line 89: Were these 15 leaves collected per plot, per individual, or in total? Does “repeated 3 times” mean the 3 plots in each treatment, or at 3 different times? Were they collected from the side facing the road or the other side, and from the top of the plant or the bottom?
Line 108: More information on sampling is needed. Does “more than 3 biological repeats” mean 3 leaves per plant? How many plants in each plot were sampled? What time of day were they sampled, and how were leaves transported and stored? Because sugar levels can fluctuate due to a number of factors, it is often more informative if starch is quantified as well.
Line 118: Information on sampling is needed.
Line 136: No description is given in the methods of how growth was measured, and no growth data is presented. If this is just a visual assessment, it should be clearly stated, such as “A visual assessment suggested that growth in the control plots was weak….” What was the condition of the plants at the beginning of the experiment? If grown in this location for 6 years, were dead branches visible before beginning the experiment?
Line 147: No description is given in the methods of how root growth was measured, and no growth data is presented. Therefore “…of roots…” should be removed.
Table 2: Are all of the parameters listed here relevant and meaningful? Usually chlorophyll fluorescence data is presented as Fv/Fm and NPQ. Results would be easier to interpret if data was presented in these standard forms.
Tables 3, 4, and 5: I assume values are means, but it is not stated whether the value that follows is standard error or standard deviation.
Line 285: “After 5-ALA treatment…” – were measurements made before and after treatment, or is this actually a comparision of treated vs. non-treated plants?
Line 388: Since exhaust fumes were not great during this time, does that not suggest that some other factor, possibly soil chemistry or water availability, is the primary stressor?
Line 411: No data was collected on the occurrence of new roots, or the probability of plant death, therefore this sentence should be removed.
Line 471: Was soil pH tested to see whether it was affected by 5-ALA treatments? Is it possible that the effects of 5-ALA treatment on mineral content are the result of pH changes, or indirect effects of the treatment on mycorrhizal activity?
Author Response
Line 47: Is there also salt applied to roadways in the winter?
Answer: It is a very nice question! Yes, when it snows in the winter, all highways here are applied with industrial salt (NaCl) to prevent traffic accidents. It is an important factor to destroy soil and impair growth of greening trees nearby. We have added the situation in the revision.
“In summer, it continuously rains almost throughout the whole June and most of July, which floods the whole asphalt road as well as the greening trees for a long time. In winter, it often snows, and a great deal of industrial salt (NaCl) is sprinkled on the road to prevent traffic accidents, which makes salination of soils and underground water”.
Line 70: It would be helpful to include a 1-2 sentence description of this species.
Answer: Thank you for your kind suggestion. We have added several sentences in the revision.
“Buxus megistophylla is an evergreen shrub species [21], which is widely planted on the roadside in urban and rural public green space, road hedgerow, ecological ditch and other related landscape construction in many regions of China [22]. It has been proposed that the species is a moderate sensitive to drought [23], chilling [24] and air pollution [25]. Yet, how to improve its stress tolerance has never been reported. ”
Line 78: Had these plants been growing in this location for 6 years, or were they planted just prior to the experiment?
Answer: The plants had been planted for six years before the experiment. We rewrote the description as follows.
“The materials selected in this experiment were Buxus megistophylla Levl. hedgerows which were constructed on the both sides of Nenjiang Road, Changzhou City, Jiangsu Province 6 years ago”.
Line 81: How many individuals were present in the 100 m long plot?
Answer: There were a great amount of plants in 100 m long plot, because the planting density was 64 cutting plantlets in a square meter when they were planted 6 years ago. When we investigated the experimental results, overcrowded plants made it difficult to dig out a plant with a complete root system from the dense hedges without destroying other plants. It is why we did not provide data about root growth. In the revision, we added a sentence as follows.
“The planting density was 64 cutting plantlets in a square meter, and the hedgerows were monthly pruned to keep 70-80 cm high during growth seasons”.
Lines 83-84: What is meant by “generally surveyed”? Please describe in detail what measurements were made and how.
Answer: Just as what we said above, too crowded hedgerow trees blocked us to dig up a plant with a complete root system. On the other hand, the hedgerow trees were regularly pruned in order to keep plants in uniform height, which blocked us to measure shoot growth. Therefore, what we could do was to compare the leaf characteristics including the sizes, the thickness, the color and chlorophyll fast fluorescence. Additionally, we could investigate plant survival between the treatment and the control. In the actual roadside investigation, we could find out the promotion of 5-ALA treatment clearly without any instruments because the difference was so significant that could be seen by the naked eye. It was why we decided to study the mechanism of 5-ALA. We randomly dug up 5-8 plants with partial roots from different plots, rinsed thoroughly, took some of leaves and root tips off and put into liquid nitrogen for quick freezing and drove back soon. After arriving in the laboratory, the frozen samples were transferred to the refrigerator at -80 ℃ waiting for physiological and biochemical analysis. The procedure has been added in the revision.
Line 89: Were these 15 leaves collected per plot, per individual, or in total? Does “repeated 3 times” mean the 3 plots in each treatment, or at 3 different times? Were they collected from the side facing the road or the other side, and from the top of the plant or the bottom?
Answer: Yes, what you said is right. There were so many plants in our experimental plots that 15 leaves were collected from different plants of each plot. “Three repeats” means 3 plots. All the mature leaves were collected from the middle of plants since the tips of shoots were pruned regularly, which were grown on the both sides of the road.
Line 108: More information on sampling is needed. Does “more than 3 biological repeats” mean 3 leaves per plant? How many plants in each plot were sampled? What time of day was they sampled, and how were leaves transported and stored? Because sugar levels can fluctuate due to a number of factors, it is often more informative if starch is quantified as well.
Answer: We collected the leaves of B. megistophylla Levl. from different plants in each plot in the morning of the 28th of July. “More than 3 biological repeats” means that we took the biological samples more than 3 times when the actual analysis was conducted. The chlorophyll fast fluorescence curves were measured in the field with the in vivo leaves, where 15 leaves from different plants and experimental plots were randomly selected. The other samples for physiological and biochemical analysis were treated by liquid nitrogen and kept in -80℃ fridge until analysis. Therefore, we think that the enzyme activity and the soluble sugar content were not affected by the external environment. In the revision, we replace “more than 3 repeats” with “3 repeats”, because some of parameter measurements such as enzyme activity analysis were biological 3 repeats, although we took technical repeats 2 times. It means that an average value was obtained from more than 3 times repeats. The way we treated our data meets the criterion of biological statistics.
Line 118: Information on sampling is needed.
Answer: The mature leaf and fine root samples used in mineral element analysis were taken back at ordinary temperature. They were washed very clearly with tap water and then deionized water, then dried and ground into fine powder. When analysis was conducted, 0.1 g dry powder was taken for digestion and measurements. Three biological repeats were set. We have modified it in the revision.
Line 136: No description is given in the methods of how growth was measured, and no growth data is presented. If this is just a visual assessment, it should be clearly stated, such as “A visual assessment suggested that growth in the control plots was weak….” What was the condition of the plants at the beginning of the experiment? If grown in this location for 6 years, were dead branches visible before beginning the experiment?
Answer: What you said is right. We could not measure the shoot growth because of monthly pruning. However, since the effect of 5-ALA treatment was so significant, one could find out the difference between the control and 5-ALA treatment by the naked eye. Actually, we were all amazed by the differences between the control and the treatment. The promotive effects are not questionable.
Line 147: No description is given in the methods of how root growth was measured, and no growth data is presented. Therefore “…of roots…” should be removed.
Answer：We could not get a complete root system from the crowded hedgerow, neither the shoot growth because of pruning, but we could judge the significant effect after 5-ALA treatment in the field investigation. Therefore, we accepted your suggestion to remove “of roots”.
Table 2: Are all of the parameters listed here relevant and meaningful? Usually chlorophyll fluorescence data is presented as Fv/Fm and NPQ. Results would be easier to interpret if data was presented in these standard forms.
Answer: The parameters in Table 2 are all significant at P = 0.05 or 0.01 level. Fv/Fm is an index often used in chlorophyll fluorescence, and NPQ is used only in modulated fluorescence measurement. M-PEA is a non-modulated fluorescence analyzer, therefore, NPQ is not suitable. We studied chlorophyll fluorescence more than a dozen years, with modulated fluorescent instrument or non-modulated fluorescent instrument. We are familiar with their principles and applications. There is no question in the aspect.
Tables 3, 4, and 5: I assume values are means, but it is not stated whether the value that follows is standard error or standard deviation.
Answer: Yes, what you said is right. The values in tables 3, 4 and 5 are the means of three biological repeats and the following values are the standard errors. I think the expression style accords with the standard of scientific articles.
Line 285: “After 5-ALA treatment…” – were measurements made before and after treatment, or is this actually a comparison of treated vs. non-treated plants?
Answer: You proposed a good question. We have revised the expression.
Line 388: Since exhaust fumes were not great during this time, does that not suggest that some other factor, possibly soil chemistry or water availability, is the primary stressor?
Answer: Maybe, but not sure. According to news reports, the pandemic of COVID-19 in China was generally alleviated in April, 2020, almost the same time when we began our experiments. After then, the traffic volume increased greatly. We can guess that the stress from the waste gas of traffic was less than the other years but not exclude its harmful effect. The exhaust pollution around hedgerows is an important aspect in our urbanization process, which needs further study.
Line 411: No data was collected on the occurrence of new roots, or the probability of plant death, therefore this sentence should be removed.
Answer: What you said may be right. We did not present data of root growth, because the plants were grown too crowded for us to dig up a complete plant with the whole root system. But we indeed qualitatively compared the roots between the control and 5-ALA treatment, and found so many differences. We think that the statement here is in Discussion, rather than Results, and, even though no precise data, we can declare the fact that 5-ALA treatment promote plant root growth in the study, which is accordant with many previous reports.
Line 471: Was soil pH tested to see whether it was affected by 5-ALA treatments? Is it possible that the effects of 5-ALA treatment on mineral content are the result of pH changes, or indirect effects of the treatment on mycorrhizal activity?
Answer: We do not determine the soil pH or mycorrhizal activity, because there is no report suggesting that 5-ALA application on plant leaves would affect soil pH or mycorrhizal activity. We may pay attention to the aspects in the further study. Thank you for the fantastic suggestion.
Reviewer 2 Report
10: Green trees? Do you mean indeciduous?
11: I can’t see the connection of stressful environments to natural soils and bad weather
18: What is a “greater” leaf colour?
34: Please clarify if this paper is about plant production or plant usage?
39-54: No citations!
42: Guest soil?
58: Please name “and so on”
77: Please describe plants more precisely. Hight, diameter, damages and conditions before treatment are missing.
87: Which leaves were measured? Please describe position of leaves within a twig.
89: It is not clear on how many plants measurements were conducted.
94: Was this done on different trees?
109, 116, 128: “more than three” is not a precise statement. Four is a very low n.
118: Which roots were used? Coarse or fine roots? Including mycorrhizal fungi?
130: Normal distribution tested? Was it allowed to use Student’s test?
134: Describe “Appearance” better
136: “Field investigations” were not described in Material and Methods
137: Description of plants before treatment is missing. Plants on control plants were weak, but on treatment plots?
140: Procedure of root analysis not described
146: In Table 1 no root data are shown!
268, 278, 304: Please show n and add p values
366: Add structure to discussion, add citations and don’t repeat information from introduction.
536: Please investigate role of mycorrhizal fungi in this case. They are known to hold back heavy metals.
Author Response
10: Green trees? Do you mean indeciduous?
Answer: Not complete. What we said here is not only a simple indeciduous tree, but any greening plant. Coincidently, B. megistophylla Levl. is an evergreen plant species. Therefore, one can consider it as any greening plant or an evergreen plant. In order to prevent misunderstanding, we replaced it with “greening trees” in the revision.
11: I can’t see the connection of stressful environments to natural soils and bad weather.
Answer: Yes, maybe you are right. We have replaced “natural soils” with “local soils”. Just as you that, as a perennial hedgerow, B. megistophylla Levl. plants are encountered many kinds of environmental stresses once they are planted on both sides of the road. On one hand, the exhaust from the heavy traffic contains a large number of pollutants such as harmful heavy metals, nitrogen oxygen compounds, which pervade into hedgerows. The soils used to plant the trees are often shortage in granular structure and fertilizers. In summer, the continuous rains cause floods of roads and trees, while the winter snow needs to apply industrial salt on road to prevent traffic accident, which will make soil salinization. All the situations are not conducive to plant growth. This is why we said that the poor soil, bad weather and traffic pollution are all stressful factors affecting B. megistophylla Levl. growth.
18: What is a “greater” leaf colour?
Answer: Yes, you proposed a very good question. We revised it as follows.
“the leaf size and thickness of B. megistophylla were significantly greater in 5-ALA treatment than those of the control, and the leaf color was also greener than the latter”.
34: Please clarify if this paper is about plant production or plant usage?
Answer: What we study is eventually for its application. 5-ALA is a natural non-protein amino acid, which we have been studying since 2000. In agricultural production, we and many other scholars at home and abroad have reported many effects of 5-ALA on improving grain yield, and enhancing product quality. More importantly, 5-ALA can improve plant stress tolerance in many aspects, which we had described in Introduction. However, whether 5-ALA can be used to improve the stress tolerance for hedgerows along highway roadsides has never been reported. After our study, we can say that 5-ALA can be used in the species of landscape trees. Actually, it has been used in garden and landscape plant management in Changzhou city since our results were known by the local government.
39-54: No citations!
Answer: Thank you for your reminder. We have added references in the paragraph.
42: Guest soil?
Answer: We used “guest soil” to represent the soil moved from elsewhere in order to improve the soil of this place. In Baidu Translation, we can find an example of “guest soil”. Additionally, “foreign soil” is also found in Baidu Translation. Should we use “foreign soil” instead of “guest soil”? Maybe both of them are OK.
58: Please name “and so on”.
Answer: 5-Aminolevulinic acid (5-ALA) is a δ-amino acid, but does not participate in protein synthesis. It acts as an essential biosynthetic precursor of all organic heterocyclic tetrapyrrole compounds, including chlorophylls, vitamin B12, heme, billins, siroheme and so on. Additionally, there are many intermediate products, which all are biosynthesized from 5-ALA as precursor. We do not need to list all of them. Therefore, we use “and so on” to represent them.
77: Please describe plants more precisely. Hight, diameter, damages and conditions before treatment are missing.
Answer: Miss Xu Yi, one of our authors, is a manager of the Changzhou Park Management Center. According to her opinion, city road hedgerows are different from the other green plants. They are planted in a very high density and pruned monthly to keep in a certain height. In our experimental hedgerows, the planting density was 64 plants (cuttings) per square meter when they were established 6 years ago, and the plant height were maintained about 70-80 cm with diameters of 1.5-2.0 cm by pruning throughout whole years. Therefore, we need not care the plant height or diameter before and after 5-ALA treatment. The most basic but important standard to assess garden plants is alive. If the plants die, the greening means failure. Healthy shoots with green leaves represent greening success. Before our experiment, the highway hedgerows were often criticized by the local leaders. It is why she asked us to test in their regions. In actual experiments, we also tested the effects of other two chemical besides 5-ALA on the hedgerow growth. Yet, one of them was not effective, and another was harmful for plant growth, whose leaves were almost all chlorosis, worse than the control. Only 5-ALA treatment exhibited a very significantly promotive effect. Miss Xu and her leaders all satisfied the experimental results, and 5-ALA has therefore been accepted as a normal technical product to apply in hedgerow management in the region. Besides B. megistophylla reported here, we also used Gardenia jasminoides Ellis as test materials. The promotive effect of 5-ALA obtained was the same. Therefore, we did not measure the height or diameter of plants before 5-ALA treatment, but we assure that the effect is real and effective.
87: Which leaves were measured? Please describe position of leaves within a twig.
Answer: We used the leaves located in the middle shoots, neither the tip nor the base. Actually, there were too many plants in each plot, and we randomly selected some of leaves to measure these parameters. Fortunately, the promotive effect of 5-ALA was so uniform that the standard errors we got were rather small in most parameters. We all satisfied the results we obtained.
89: It is not clear on how many plants measurements were conducted.
Answer: We spayed 5-ALA solution in a large area hedgerow, and there were so many plants that we could selected them freely. Additionally, we took back more than a dozen plants both from the control and 5-ALA treatment for physiological and biochemical analysis. We believe that our sampling method conforms to the field test rules.
94: Was this done on different trees?
Answer: Yes, you are right. We used leaves from different trees and different plots. Total 15 leaves were used to measure the chlorophyll rapid fluorescence and calculate the fluorescence parameters.
109, 116, 128: “more than three” is not a precise statement. Four is a very low n.
Answer: Thank you very much. We have modified the statement as follows. However, we do not think that three repeats are too low but standard.
118: Which roots were used? Coarse or fine roots? Including mycorrhizal fungi?
Answer: The fine roots were used in analysis. But we did not consider mycorrhizal fungi, they were not included in our research. Maybe in the further work, we can pay attention to mycorrhizal fungi.
130: Normal distribution tested? Was it allowed to use Student’s test?
Answer: Yes. Since there were only a control and a treatment, Student’s test could be used in our work.
134: Describe “Appearance” better.（√）
Answer: We could not describe “appearance” more detailed. From the external appearance, one could see that the plants in 5-ALA treated plots grew much better than the control. All membranes of our group agreed the obvious effect when we stood by the roadside of the experimental plots. Whether the plant survival, leaf color, shoot health or new root occurrence, 5-ALA treatment was much better than the control. We have modified our description slightly in the revision.
136: “Field investigations” were not described in Material and Methods.（√）
Answer: Yes, we did not introduce specific method for field investigation, because the difference between the control and 5-ALA treatment was so significant that one could find out by the naked eye. What is more important we thought is to study the mechanism by which 5-ALA promoted plant growth under such environmental condition.
137: Description of plants before treatment is missing. Plants on control plants were weak, but on treatment plots?
Answer: Generally, the plants before our experiment were not very good. After all, they had been grown for 6 years. Why Miss Xu asked us to test was because they grew not too well. She and her leaders satisfied with our experimental results of 5-ALA treatment, but not the other two chemicals. This means that the other two chemicals were not effective, even worse than the control. Yet, all experimental plots were designed randomly, and the plants before treatment theoretically were uniform. On the other hand, the area of each plot with a great number of plants was enough large, we believed that the effect of 5-ALA reliable.
140: Procedure of root analysis not described.
Answer: Just as we said above, we could not obtain a plant with a complete root system in the overcrowded hedgerow unless we dug up a lot of plants, which would destroy the greening belt. We thought that the effect was so significant that we could assess even without a complete root system. In actual investigation, we dug up 5-8 plants with partial roots in one plot. From them, we found that some roots in the control were browned, rotted, with less new occurrence, while that in 5-ALA treatment grew much better than the control. Since we had no complete data about roots, partial data cannot be shown in the formal article.
46: In Table 1 no root data are shown!
Answer: Yes, you are right. We delete the description about root growth in the text of revision.
268, 278, 304: Please show n and add p values（√）
Answer: I am sorry. I do not understand what you suggested here. We had added P values in the footnotes of the three tables. Should we put P in the table titles? No, I do not think so. Sorry for me to say so because I have seldom seen like that before.
366: Add structure to discussion, add citations and don’t repeat information from introduction.（√）
Answer：What you said is right. We will add more citations in the discussion and avoid to repeat information in introduction. However, a few repeats may be permitted between Introduction and Discussion. We have deleted several sentences in the Discussion.
536: Please investigate role of mycorrhizal fungi in this case. They are known to hold back heavy metals.（√）
Answer: What you suggested may be right. However now, we had not prepared materials for mycorrhizal fungi study, therefore, we may study them according to your suggestion in the further. Thank you very much.
Reviewer 3 Report
Tha paper "Effect of 5-aminolevulinic acid (5-ALA) on leaf chlorophyll fast fluorescence characteristics and the mineral element content of Buxus megistophylla grown alongside urban road" by H. Yang et al. presents an interesting results concerning an influense of low concentration of non-proteinogenic amino acid to increase total tolerance of a bosxtrees which are growing under negative abiotic environments. Generally, this work has been made in the best traditions of plant physiology and I can evaluate it well. But I have got some questions that are critical for main conclusion:
- "Materials and Methods", "Determination of the activities of antioxidant enzymes in leaves and roots" - please descride in detail all of these procedures, because there are some modifications of AOX enzymes which have to be determined clearly.
- "Results", "Effects of 5-ALA treatment on the content of soluble sugar and free proline in B. megistrophylla leaves" - the authors have measured free proline as a factor of antistress action in plants, I mean?! Based on the results presented, we cannot see a quite difference between control and variant (Table 3). In this regard, I strongly recommend to expand estimation of amino acid content: for example, check serine, arginine, lysine and valine. All these free amino acids are known to participate in increasing of total resistance of plants to abiotic stress factors.
- Table 4 - SOD activities are not significant between controal and 5-ALA action. Please make it in mind to conclude all AOX enzymes.
Author Response
Comment and Answer
Reviewer 3
The paper "Effect of 5-aminolevulinic acid (5-ALA) on leaf chlorophyll fast fluorescence characteristics and the mineral element content of Buxus megistophylla grown alongside urban road" by H. Yang et al. presents an interesting result concerning an influence of low concentration of non-proteinogenic amino acid to increase total tolerance of a bosxtrees which are growing under negative abiotic environments. Generally, this work has been made in the best traditions of plant physiology and I can evaluate it well. But I have got some questions that are critical for main conclusion:
Answer: Thank you for your kind comments. We have been studying 5-ALA application in agroforestry production since 2000. However, there are much work need to do. We should work harder in the future.
Question:
"Materials and Methods", "Determination of the activities of antioxidant enzymes in leaves and roots" - please describe in detail all of these procedures, because there are some modifications of AOX enzymes which have to be determined clearly.
Answer: We would have modified the paper as your suggestion. However, the procedures for enzyme activity measurements have been reported countless times, the main processes are unchanged although they are often modified slightly. Therefore, we do not want to add several paragraphs. Even this, we do not think they will affect our results. Please forgive us.
Question:
"Results", "Effects of 5-ALA treatment on the content of soluble sugar and free proline in B. megistrophylla leaves" - the authors have measured free proline as a factor of antistress action in plants, I mean?! Based on the results presented, we cannot see a quite difference between control and variant (Table 3). In this regard, I strongly recommend to expand estimation of amino acid content: for example, check serine, arginine, lysine and valine. All these free amino acids are known to participate in increasing of total resistance of plants to abiotic stress factors.
Answer: Thank you for your good suggestions. I think we could obtain results as you predicted, the content of many kinds of free amino acids besides proline would be found different between the control and 5-ALA. From Table 5, the extremely significant increase of N in the roots and leaves in 5-ALA treatment plants implied that the amino acid content must be increased greatly. However, what is new in our work is that we found that 5-ALA treatment greatly affected the content of many mineral elements instead of amino acids. Therefore, we would detect the amino acids content in the next time, but not this time. Thank you again. Proline has been suggested as the most important amino acid related to plant stress tolerance, we select to measure it here.
Suggestion:
Table 4 - SOD activities are not significant between control and 5-ALA action. Please make it in mind to conclude all AOX enzymes.
Answer: Sorry, sir. I do not agree with what you said here. From Table 4, we can see that the SOD activities are significant at P = 0.05 level between the control and 5-ALA treatment, although the differences do not reach P = 0.01 level. We checked our primary data, and found not wrong here. Therefore, we can say that 5-ALA promotes SOD activity in B. megistophylla. The similar effect has been reported in many other species. Anyway, we thank you very much.
Round 2
Reviewer 1 Report
Thank you to the authors for considering earlier comments, and providing thoughtful revisions and responses. It is interesting that after much study, the mode of action of 5-ALA is only partially known. This study has focused on foliar application and responses, yet results suggest some effects on the root system which is very interesting! It would be interesting to know whether the effects are primary via shoot-to-root transport or applied 5-ALA washing off leaves and into soil, or secondary by within-plant signaling or influences on the soil microbiome. There are so many stresses to the plants in this study as the authors mention (salinity, seasonal flooding, likely seasonal drought, heavy metals, likely high pH, disrupted photoperiod, as well as automobile exhaust) so there may be several mechanisms of 5-ALA action.
The experimental design and and sampling are much more clear, thanks to authors revisions to the methods section. This has answered many of my questions and comments from the earlier version. Some minor English editing is noted in the attached file. This is a very nice study and interesting work!
Comments for author File: 
Comments.pdf
Author Response
Dear sir,
Thanks a lot for all your suggestions to our work. Most of notes you made in the attached file have been revised as you suggested. Thank you very much.
Reviewer 2 Report
11: I guess, they are suffering from polluted soils und maybe higher temperatures/less water supply. This is not the same as local soil and bad weather.
35: You described in the paper usage of plants and not production of plants.
90 Constructed? I would prefer “grown”.
146: At first cleaned with tap water and than with deionized water?
157: Please describe statistical analysis more precisely.
164: Even if its visible by naked eye, a rating procedure is necessary to describe the plants before and after applying 5-ALA. Description before treatment is completely missing.
289, 298: Please show n
384: Add structure to discussion. First page sounds more like an introduction than a discussion.
Author Response
Dear Sir,
Thank you very much. The follows are our answers to your kind suggestions. If there is any question, please let me know.
11: I guess, they are suffering from polluted soils and maybe higher temperatures/less water supply. This is not the same as local soil and bad weather.
Answer: Thank you for your reminding. Yes, there are many factors which may affect plant growth along two sides of the highway roads. However, high temperature/less water, which you guessed, was not the case in summer of 2020, because it rained almost the whole month of July and the highest temperature did not exceeded 32℃. What we mentioned in the abstract is general situation, rather than a special year. We cannot exclude any of them.
35: You described in the paper usage of plants and not production of plants.
Answer: What you said may be right. However, I do not think our description is wrong. In the last sentence of the abstract, we wrote that “These provide a theoretical basis for its application in city road landscaping plant production”. Do you mean we should delete “production”? I do not think it necessary. 5-ALA can be used in plants, but at the end, it can be used in plant production. Which is wrong?
90 Constructed? I would prefer “grown”.
Answer：Thanks a lot. We accept as you suggested.
146: At first cleaned with tap water and then with deionized water?
Answer: Yes, we washed them with tap water and then deionized water. In order clearer, we added “respectively” in the revision.
157: Please describe statistical analysis more precisely.
Answer: Thank you very much. However, we think what we wrote in the paragraph is correct. Nevertheless, we revised P into italic style.
164: Even if its visible by naked eye, a rating procedure is necessary to describe the plants before and after applying 5-ALA. Description before treatment is completely missing.
Answer: I am sorry to say that I cannot describe the plants before 5-ALA application. Firstly, what we focused on is the difference between the treatment and the control, rather than before and after application. Secondly, the hedgerows had grown there for 6 years, which were pruned every month during growth season to maintain height. No one can tell the real effects before and after treatment. Thirdly, maybe the most importantly, I did not go to the testing site in the late of April because of pandemic Covid-19 limit. At that time, the mobility in our country was very difficult. The people who were permitted to go out of campus and participate the experiments were as few as possible. What I can do was guiding the experiment by phone and asking them to design the plots large enough. Fortunately, there are several kilometres of hedgerows we could choice for the experiment and, the experimental effect was so significant that we are all excited up to now. We should put our eyes on the general effect but not the small detail. Do you agree with us?
289, 298: Please show n
Answer: Thank you very much. We have added the repeat times in the notes of all tables.
384: Add structure to discussion. First page sounds more like an introduction than a discussion.
Answer：Thank you very much. However, I’d like to write a discussion in present style.
Up to now, I have published papers near 200 in Chinese or English. And I have been teaching students how to write a research article more than twenty years. I also review many submitted papers for dozens of journals in China and abroad very year. I was awarded the title of “Review Expert with Outstanding Contribution” two times by a famous journal. Therefore, I am familiar with how to prepare an article including discussion. In some aspect, a discussion is similar with an introduction. Both of them are argumentation style, which need many evidences to support authors’ opinions. However, an introduction is the beginning or cause of the scientific story, while a discussion is the story after scientific research. Therefore, there may be some overlapping in two sections of an article. If it is necessary, a little overlapping is permitted, although we should limit it as possible as we can.
Every time when we prepare our article, I am very careful to notice every detail as possible as I can, although sometimes I also make mistakes, which is unavoidable. In present work, I think it is suitable. I want to thank the review experts for their rigorous and earnest work, because I am also rigorous and earnest to review the other papers. Thank you.
Reviewer 3 Report
Thanks for your replies. No comments more.
Author Response
Dear Sir,
I have read your comments on our work. Thank you for your suggestions. We will do the best to improve our manuscript until it can be accepted for publish. Thank a lot.
Best reagrds
Yours Sincerely
Prof. Liangju Wang
April 18, 2021
