24-Epibrassinolide Simultaneously Stimulates Photosynthetic Machinery and Biomass Accumulation in Tomato Plants under Lead Stress: Essential Contributions Connected to the Antioxidant System and Anatomical Structures
and Allan Klynger da Silva Lobato 1,*Round 1
Reviewer 1 Report
Dear Authors,
The introduction is a good reflection of the current state of research. The aim of the work and research hypotheses are clearly stated. However, in the case of the methodology and the description of the research results, the article should be thoroughly revised. The methodology section does not clearly describe when the tests of individual parameters were performed. Were these measurements repeated during the experiment, or were they performed once? The method of applying the factors of experience is also not entirely clear. Please describe its model more clearly. There is also a lack of information on how many repetitions the individual analyzes were carried out (in a sample or in a pot?).
My next question is why were such factor concentrations chosen and what were the guidelines behind the choice of measurement dates?
How much was the light intensity in the greenhouse different from that when the physiological parameters were measured? Was the greenhouse insolated by sunlight (daylight) or by artificial light sources in a fixed photoperiod?
Please indicate when chlorophyll fluorescence and gas exchange measurements were made? At the end of the experience? After each application? How many replications? Were the analyzes of anatomical variables of roots and leaves carried out after all treatments before the end of the experiment, or perhaps cyclically? The other analyzes are similar. When were they made and why?
In the case of the methodology, there is also no description of the method of analyzing micro and macro elements, which are described in the research results.
Please arrange the order of the presented research in the order indicated in the methodology. The current provision introduces quite a lot of confusion when reading the manuscript and indicates a general inconsistency in the content of the work. To sum up, the methodology contains a lot of information, but it is not written in an understandable way and does not coincide with the results section.
Research results - there are no references to tables and figures in the text, which makes the reception of the data difficult. As mentioned earlier, the results are not in the order of the methodology. Generally, the entire text is not prepared according to the journal's guidelines, so please also reformat it.
The discussion is well prepared and detailed. The conclusions are supported by the research results, but also difficult to read, so I recommend editing them.
In general, the research topic discussed by the authors is cognitively important and interesting. This research also has the potential to become research implemented in agricultural practice. However, the manuscript itself needs to be improved.
Author Response
Dear reviewers and editor linked to manuscript # agronomy-1825920:
We are submitting the revised manuscript to the journal submission site for your review.
As instructed in your e-mail on 16 Jul 2022, we have carefully considered all the reviewer’s comments and fully addressed them in the revised manuscript. Our responses to each specific reviewer comments are as follows (red into manuscript):
General comments from Managing Editor: Your manuscript has now been reviewed by experts in the field. Please find your manuscript with the referee reports at this link:
https://susy.mdpi.com/user/manuscripts/resubmit/5c587fadfe50f9b9c59a865debfeac8c
Please revise the manuscript according to the referees' comments and upload the revised file within 7 days.
Decision: Major Revisions
Holley Liu
Assistant Editor
Agronomy
General comments from Reviewer 1: Dear Authors,
The introduction is a good reflection of the current state of research. The aim of the work and research hypotheses are clearly stated. However, in the case of the methodology and the description of the research results, the article should be thoroughly revised. The methodology section does not clearly describe when the tests of individual parameters were performed. Were these measurements repeated during the experiment, or were they performed once? The method of applying the factors of experience is also not entirely clear. Please describe its model more clearly. There is also a lack of information on how many repetitions the individual analyzes were carried out (in a sample or in a pot?). In general, the research topic discussed by the authors is cognitively important and interesting. This research also has the potential to become research implemented in agricultural practice. However, the manuscript itself needs to be improved.
Reviewer 1: My next question is why were such factor concentrations chosen and what were the guidelines behind the choice of measurement dates?
Authors: These informations were included in manuscript (below):
“Pb and EBR concentrations were chosen according to studies of Guedes et al. [32] and Maia et al [33], respectively.”
“This study aimed to verify whether the exogenous application of EBR can preserve tomato plants from oxidative damages caused by excess Pb, evaluating the responses associated with leaf anatomy, antioxidant metabolism, photosynthetic apparatus, and biomass.”
Reviewer 1: How much was the light intensity in the greenhouse different from that when the physiological parameters were measured? Was the greenhouse insolated by sunlight (daylight) or by artificial light sources in a fixed photoperiod?
Authors: These informations were inserted (below):
“The study was conducted in a greenhouse illuminated with sunlight, but with temperature and humidity controlled. The minimum, maximum, and median temperatures were 23.3, 29.2, and 25.5 °C, respectively. The relative humidity during the experimental period varied between 60% and 80% and photoperiod of 12/12.”
“Gas exchange was evaluated in all plants under a constant CO2 concentration (390 μmol mol-1 CO2), photosynthetically active radiation (800 μmol photons m-2 s-1), air-flow rate (300 µmol s-1), and temperature (28ºC) in the test chamber between 10:00 and 12:00 h.”
Reviewer 1: Please indicate when chlorophyll fluorescence and gas exchange measurements were made? At the end of the experience? After each application? How many replications? Were the analyzes of anatomical variables of roots and leaves carried out after all treatments before the end of the experiment, or perhaps cyclically? The other analyzes are similar. When were they made and why?
Authors: Informations connected to data collection and number of replicates used in this research has been added and inserted in all legends of tables and figures (below):
“On day 30 of the experiment, physiological and morphological parameters were measured for all plants, and tissues were harvested for anatomical, biochemical and nutritional analyses.”
“Five replicates for each one of the four treatments were conducted, used in the experiment a total of 20 experimental units, with three plants in each unit.”
Reviewer 1: In the case of the methodology, there is also no description of the method of analyzing micro and macro elements, which are described in the research results.
Authors: This methodology was inserted in manuscript (below):
2.6. Determination of Pb and nutrients
Milled samples (100 mg) of root, stem and leaf tissues were pre-digested in conical tubes (50 mL) with 2 ml of sub boiled HNO3. Subsequently, 8 ml of a solution containing 4 ml of H2O2 (30% v/v) and 4 ml of ultra-pure water were added and transferred to a Teflon digestion vessel in agreement with Paniz et al. [33]. Determination of Pb, Mg, K, Ca, Cu, Zn and Mn was performed using an inductively coupled plasma mass spectrometer (model ICP-MS 7900; Agilent).
Reviewer 1: Please arrange the order of the presented research in the order indicated in the methodology. The current provision introduces quite a lot of confusion when reading the manuscript and indicates a general inconsistency in the content of the work. To sum up, the methodology contains a lot of information, but it is not written in an understandable way and does not coincide with the results section.
Authors: Methodology was put in order with results section.
Reviewer 1: Research results - there are no references to tables and figures in the text, which makes the reception of the data difficult. As mentioned earlier, the results are not in the order of the methodology. Generally, the entire text is not prepared according to the journal's guidelines, so please also reformat it.
Authors: All tables and figures were mentioned in results section and methodology was put in order.
Reviewer 1: The discussion is well prepared and detailed. The conclusions are supported by the research results, but also difficult to read, so I recommend editing them.
Authors: Thank you very much. This topic was improved.
General comments from Reviewer 2: The authors evaluate the application of EBR in tomato plants subjected to lead stress. They analyze different variables related to the antioxidant system and stress biomarkers, mineral content, photosynthesis and fluorescence, among others, which makes this work very complete. The results obtained are excellent and forceful, since they demonstrate the positive effects of the EBR application. However, there are some comments about the work.
Reviewer 2: The experimental work is carried out in a very short period, ideally it should be carried out in a complete cycle, this would allow determining if lead accumulates in tomato fruits.
Authors: Thank you very much for the suggestions (treatment time and fruit contamination), in our next research we will work on this objective (if Pb can accumulate in the fruit).
Reviewer 2: Check the format of references cited in the text.
Authors: All references were checked and corrected.
Reviewer 2: Standardize the use of abbreviations. In some cases the authors use "Pb2+ + 100nM EBR treatment" and in others "Pb + EBR".
Authors: We appreciate the suggestion and dedication of reviewer 2 during the review process of this manuscript, but in our opinion it would be too monotonous to use only one form.
Reviewer 2: Line 165. What was the reason for using PbCl2 as a source of Pb?
Authors: This molecule (PbCl2) will provide the target element (Pb) and an essential element (Cl) in low/irrelevant concentration.
Reviewer 2: Line 177. What was the reason for determining the fluorescence under light and not in the dark?
Authors: We used leaves that grew under light, but at the moment of measurement they were kept for 30 minutes in the dark. The sentence has been improved and clarified (below):
“The chlorophyll fluorescence was measured in fully expanded leaves under light. Preliminary tests determined that the acropetal third of leaves in the middle third of the plant and that adapted to the dark for 30 min yielded the greatest Fv/Fm ratio. Therefore, this part of the plant was used for measurements. The intensity and duration of the saturation light pulse were 7500 µmol m–2.s–1 and 0.7 s, respectively.”
Reviewer 2: Line 180. Why were these values selected?
Authors: These values were defined after preliminary tests.
Reviewer 2: Line 333. The authors say "compared to the Pb2+ + 0nM EBR treatment." Is this correct?
Authors: This sentence was corrected “compared to the Pb treatment”.
Reviewer 2: Line 377. Explain in more detail how EBR minimizes metal translocation.
Authors: This sentence was improved and explained these results (below):
“EBR plays an important role in signalization, regulation, and differentiation of the root tissue, inducing increments in root structures after the application of this steroid, especially in RET, RDT, and RMD, being these results clearly connected to protection of this organ against the deleterious effects occasioned by the Pb [57,58]”
Reviewer 2: Line 429. The authors say "EBR application relieved the damage generated by Pb excess in F0, Fv e Fv/Fm." However, they do not explain how this is achieved.
Authors: This sentence was improved and explained these results (below):
“EBR application relieved the damage generated by Pb excess in F0, Fv, and Fv/Fm. Pb harms the efficiency of PSII photochemical reaction and electron transport chain, resulting in decreases in Fv/Fm, which may also be connected to impaired QA oxidation, further causing a decrease in electron transport from PSII to PSI [77]. The variable Fv/Fm is related to the functional state of the oxygen evolution complex and can be used as a sensitive indicator of photosynthetic performance, and when found at low levels, together with higher levels of F0, it indicates extensive photoinhibition due to environmental stresses [78]. In contrast, EBR can increase Fv and Fv/Fm and reduce F0 values, diminishing the photoinhibition effects and decreasing the dissipation of excitation energy in the antennas of photosystem II [58].”
We thank you again for your time and effort in handling and reviewing our manuscript and we are looking forward to hearing from you.
Sincerely yours,
Allan Klynger da Silva Lobato
Professor / Universidade Federal Rural da Amazônia
+55 91 993134006
Reviewer 2 Report
The authors evaluate the application of EBR in tomato plants subjected to lead stress. They analyze different variables related to the antioxidant system and stress biomarkers, mineral content, photosynthesis and fluorescence, among others, which makes this work very complete. The results obtained are excellent and forceful, since they demonstrate the positive effects of the EBR application. However, there are some comments about the work.
General comments:
-The experimental work is carried out in a very short period, ideally it should be carried out in a complete cycle, this would allow determining if lead accumulates in tomato fruits.
-Check the format of references cited in the text.
-Standardize the use of abbreviations. In some cases the authors use "Pb2+ + 100nM EBR treatment" and in others "Pb + EBR".
Specific comments:
Line 165. What was the reason for using PbCl2 as a source of Pb?
Line 177. What was the reason for determining the fluorescence under light and not in the dark?
Line 180. Why were these values selected?
Line 333. The authors say "compared to the Pb2+ + 0nM EBR treatment." Is this correct?
Line 377. Explain in more detail how EBR minimizes metal translocation.
Line 429. The authors say "EBR application relieved the damage generated by Pb excess in F0, Fv e Fv/Fm." However, they do not explain how this is achieved.
Author Response
Dear reviewers and editor linked to manuscript # agronomy-1825920:
We are submitting the revised manuscript to the journal submission site for your review.
As instructed in your e-mail on 16 Jul 2022, we have carefully considered all the reviewer’s comments and fully addressed them in the revised manuscript. Our responses to each specific reviewer comments are as follows (red into manuscript):
General comments from Managing Editor: Your manuscript has now been reviewed by experts in the field. Please find your manuscript with the referee reports at this link:
https://susy.mdpi.com/user/manuscripts/resubmit/5c587fadfe50f9b9c59a865debfeac8c
Please revise the manuscript according to the referees' comments and upload the revised file within 7 days.
Decision: Major Revisions
Holley Liu
Assistant Editor
Agronomy
General comments from Reviewer 1: Dear Authors,
The introduction is a good reflection of the current state of research. The aim of the work and research hypotheses are clearly stated. However, in the case of the methodology and the description of the research results, the article should be thoroughly revised. The methodology section does not clearly describe when the tests of individual parameters were performed. Were these measurements repeated during the experiment, or were they performed once? The method of applying the factors of experience is also not entirely clear. Please describe its model more clearly. There is also a lack of information on how many repetitions the individual analyzes were carried out (in a sample or in a pot?). In general, the research topic discussed by the authors is cognitively important and interesting. This research also has the potential to become research implemented in agricultural practice. However, the manuscript itself needs to be improved.
Reviewer 1: My next question is why were such factor concentrations chosen and what were the guidelines behind the choice of measurement dates?
Authors: These informations were included in manuscript (below):
“Pb and EBR concentrations were chosen according to studies of Guedes et al. [32] and Maia et al [33], respectively.”
“This study aimed to verify whether the exogenous application of EBR can preserve tomato plants from oxidative damages caused by excess Pb, evaluating the responses associated with leaf anatomy, antioxidant metabolism, photosynthetic apparatus, and biomass.”
Reviewer 1: How much was the light intensity in the greenhouse different from that when the physiological parameters were measured? Was the greenhouse insolated by sunlight (daylight) or by artificial light sources in a fixed photoperiod?
Authors: These informations were inserted (below):
“The study was conducted in a greenhouse illuminated with sunlight, but with temperature and humidity controlled. The minimum, maximum, and median temperatures were 23.3, 29.2, and 25.5 °C, respectively. The relative humidity during the experimental period varied between 60% and 80% and photoperiod of 12/12.”
“Gas exchange was evaluated in all plants under a constant CO2 concentration (390 μmol mol-1 CO2), photosynthetically active radiation (800 μmol photons m-2 s-1), air-flow rate (300 µmol s-1), and temperature (28ºC) in the test chamber between 10:00 and 12:00 h.”
Reviewer 1: Please indicate when chlorophyll fluorescence and gas exchange measurements were made? At the end of the experience? After each application? How many replications? Were the analyzes of anatomical variables of roots and leaves carried out after all treatments before the end of the experiment, or perhaps cyclically? The other analyzes are similar. When were they made and why?
Authors: Informations connected to data collection and number of replicates used in this research has been added and inserted in all legends of tables and figures (below):
“On day 30 of the experiment, physiological and morphological parameters were measured for all plants, and tissues were harvested for anatomical, biochemical and nutritional analyses.”
“Five replicates for each one of the four treatments were conducted, used in the experiment a total of 20 experimental units, with three plants in each unit.”
Reviewer 1: In the case of the methodology, there is also no description of the method of analyzing micro and macro elements, which are described in the research results.
Authors: This methodology was inserted in manuscript (below):
2.6. Determination of Pb and nutrients
Milled samples (100 mg) of root, stem and leaf tissues were pre-digested in conical tubes (50 mL) with 2 ml of sub boiled HNO3. Subsequently, 8 ml of a solution containing 4 ml of H2O2 (30% v/v) and 4 ml of ultra-pure water were added and transferred to a Teflon digestion vessel in agreement with Paniz et al. [33]. Determination of Pb, Mg, K, Ca, Cu, Zn and Mn was performed using an inductively coupled plasma mass spectrometer (model ICP-MS 7900; Agilent).
Reviewer 1: Please arrange the order of the presented research in the order indicated in the methodology. The current provision introduces quite a lot of confusion when reading the manuscript and indicates a general inconsistency in the content of the work. To sum up, the methodology contains a lot of information, but it is not written in an understandable way and does not coincide with the results section.
Authors: Methodology was put in order with results section.
Reviewer 1: Research results - there are no references to tables and figures in the text, which makes the reception of the data difficult. As mentioned earlier, the results are not in the order of the methodology. Generally, the entire text is not prepared according to the journal's guidelines, so please also reformat it.
Authors: All tables and figures were mentioned in results section and methodology was put in order.
Reviewer 1: The discussion is well prepared and detailed. The conclusions are supported by the research results, but also difficult to read, so I recommend editing them.
Authors: Thank you very much. This topic was improved.
General comments from Reviewer 2: The authors evaluate the application of EBR in tomato plants subjected to lead stress. They analyze different variables related to the antioxidant system and stress biomarkers, mineral content, photosynthesis and fluorescence, among others, which makes this work very complete. The results obtained are excellent and forceful, since they demonstrate the positive effects of the EBR application. However, there are some comments about the work.
Reviewer 2: The experimental work is carried out in a very short period, ideally it should be carried out in a complete cycle, this would allow determining if lead accumulates in tomato fruits.
Authors: Thank you very much for the suggestions (treatment time and fruit contamination), in our next research we will work on this objective (if Pb can accumulate in the fruit).
Reviewer 2: Check the format of references cited in the text.
Authors: All references were checked and corrected.
Reviewer 2: Standardize the use of abbreviations. In some cases the authors use "Pb2+ + 100nM EBR treatment" and in others "Pb + EBR".
Authors: We appreciate the suggestion and dedication of reviewer 2 during the review process of this manuscript, but in our opinion it would be too monotonous to use only one form.
Reviewer 2: Line 165. What was the reason for using PbCl2 as a source of Pb?
Authors: This molecule (PbCl2) will provide the target element (Pb) and an essential element (Cl) in low/irrelevant concentration.
Reviewer 2: Line 177. What was the reason for determining the fluorescence under light and not in the dark?
Authors: We used leaves that grew under light, but at the moment of measurement they were kept for 30 minutes in the dark. The sentence has been improved and clarified (below):
“The chlorophyll fluorescence was measured in fully expanded leaves under light. Preliminary tests determined that the acropetal third of leaves in the middle third of the plant and that adapted to the dark for 30 min yielded the greatest Fv/Fm ratio. Therefore, this part of the plant was used for measurements. The intensity and duration of the saturation light pulse were 7500 µmol m–2.s–1 and 0.7 s, respectively.”
Reviewer 2: Line 180. Why were these values selected?
Authors: These values were defined after preliminary tests.
Reviewer 2: Line 333. The authors say "compared to the Pb2+ + 0nM EBR treatment." Is this correct?
Authors: This sentence was corrected “compared to the Pb treatment”.
Reviewer 2: Line 377. Explain in more detail how EBR minimizes metal translocation.
Authors: This sentence was improved and explained these results (below):
“EBR plays an important role in signalization, regulation, and differentiation of the root tissue, inducing increments in root structures after the application of this steroid, especially in RET, RDT, and RMD, being these results clearly connected to protection of this organ against the deleterious effects occasioned by the Pb [57,58]”
Reviewer 2: Line 429. The authors say "EBR application relieved the damage generated by Pb excess in F0, Fv e Fv/Fm." However, they do not explain how this is achieved.
Authors: This sentence was improved and explained these results (below):
“EBR application relieved the damage generated by Pb excess in F0, Fv, and Fv/Fm. Pb harms the efficiency of PSII photochemical reaction and electron transport chain, resulting in decreases in Fv/Fm, which may also be connected to impaired QA oxidation, further causing a decrease in electron transport from PSII to PSI [77]. The variable Fv/Fm is related to the functional state of the oxygen evolution complex and can be used as a sensitive indicator of photosynthetic performance, and when found at low levels, together with higher levels of F0, it indicates extensive photoinhibition due to environmental stresses [78]. In contrast, EBR can increase Fv and Fv/Fm and reduce F0 values, diminishing the photoinhibition effects and decreasing the dissipation of excitation energy in the antennas of photosystem II [58].”
We thank you again for your time and effort in handling and reviewing our manuscript and we are looking forward to hearing from you.
Sincerely yours,
Allan Klynger da Silva Lobato
Professor / Universidade Federal Rural da Amazônia
+55 91 993134006
Round 2
Reviewer 1 Report
The Authors revised the manuscript and responded to comments. I recommend publishing in the current form.
