Exogenous Melatonin Improves the Quality Performance of Rice under High Temperature during Grain Filling
, Yun Zhu 4,†, Qianfeng Li 1,2,3,4, Lin Zhang 1,2,3,4, Dongsheng Zhao 1,2,3,4, Lichun Huang 1,2,3,4, Changquan Zhang 1,2,3,4 and Qiaoquan Liu 1,2,3,4,*
Round 1
Reviewer 1 Report
Growing conditions (substrate, number of plants per pot etc. ) need to be presented in section of materials and methods.
Titles of the sections need to be formatted uniformly.
Line 255 - "short" should be changed with "long".
Few style mistakes appear in the manuscript, e.g. line 314-315. Thus the manuscript should be revised for eliminating such an mismatches.
Author Response
Dear reviewer,
Many thanks for your constructive suggestions and recognition of our research. We have made serious revisions to our manuscript.
Point 1: Titles of the sections need to be formatted uniformly..
Response 1: All titles of the sections have been formatted uniformly.
Point 2: Line 255 - "short" should be changed with "long"..
Response 2: The word- “short” on line 255 has been changed with “long”.
Point 3: Few style mistakes appear in the manuscript, e.g. line 314-315. Thus, the manuscript should be revised for eliminating such a mismatch..
Response 3: The grammatical and formatting errors in the manuscript have been carefully corrected, including the lines from 314 to 315.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The work is aimed to analyse the effects of exogenous melatonin on growth, yield, and quality traits of one japonica rice variety, under controlled conditions, focusing on the starch synthesis, in response to high temperature during the grain filling period. The manuscript is original for melatonin use in plants, relevant and very well written. Methodology is adequate (but some points need clarificaton), Results are clearly stated and the Conclusions are supported by the data. Despite being a study at the plant and organ scale, with a single rice variety, and under controlled environmental conditions, the results obtained are of interest to readers of an agronomy journal (more over if seed yield and seed weight data are included), given the impact of thermal stress on global plant production.
Some points to be improved/clarified are listed below.
L 22 ‘resistance properties’ explain resistance to what (stomatic? mechanic? of membranes?)
L 22-23: ‘Although melatonin could not alleviate the reduction of rice yield caused by continuous high temperature’ No data on seed yield or seed weight are shown in the manuscript. As they are both very important outputs for agronomy, I encourage authors to show it (or provide a citation if those data has already been published). Otherwise, mention of seed yield should be removed from the Abstract.
L 102: ‘Subsequent studies have found that melatonin is also common in higher plants’. Give more context about sites in the plant where the endogenous melatonin is synthetised (roots?, leaves? meristematic tissues?)
L 107: ‘Besides, exogenous melatonin has been shown to confer plant tolerance to a variety of environmental stresses’ Give more context about what is the route of entry of exogenous melatonin to the plant, and how is it transported within the plant.
L 116-122: Inform the growing conditions (controlled, growth chambers) and moderate the significanceof the results, avoiding to scale from individual and controlled plants to rice production in farming systems.
L 127: ‘Before flowering, all rice materials were cultivated’ Give more context about number and size of pots/containers, soil/subtrate characteristics, irrigation, fertilization (nutrient solution applied throughout the cycle?), plant poulation density (plants/m2), and temperature of soil (as a proxy of root temperature). All these growing conditions have demostrated to modulate the heat stress response.
L 131-132: ‘The other environmental conditions in the artificial climate chamber were the same as those for the plants cultivated at normal temperature.’ Heated chambersusually modifies both, temperature and air humidity, affecting vapour pression deficit (VPD) between leaf and air. Please, include calculation of VPD for both chambers (normal and heated) from temperature and air humidity values .
L 133-135: ‘500 mL of 200 μmol L−1 exogenous melatonin (Sigma, USA) was used to treat rice materials planted’…’exogenous substances were sprayed three times every 5 days. ‘ It is not clear the whole melatonin dosis applied, please express itper plant or per m2. It was sprayed every 5 days during the whole grain filling period? (up to physiologycal maturity?) Also, justify the dosis and concentration used (previous data, preliminary assay?). For the Discussion section, are these application volumes feasible in extensive farming systems? Is there any obvious environmental risk of releasing unusual concentrations of melatonin into the environment?
L 137: ‘Flower marking’ Flower tagging could be better.
L 152: 2.4. ‘Preparation of Seed Samples’ Describe the methodology for chalkiness determination.
L 192-193: ‘three biological replicates’ of ‘three independent experiments’ What was the experimental unit? Individual plant? Individual pot with several plants? (detail what was the observation unit that received the treatments independently). Was the whole experiment repeated three times? Was each experiment considered as blocks? Please explain the experimental design.
L 207-209: Comparing C and MT treatments, melatonin enhances SOD and CAT activities under normal temperature. Why? Is it an indicator of oxidative stress caused by melatonin per se? Could spraying melatonin on non-stressed plants cause a seed yield penalty? Explain in the Results and expand it in the Discussion.
Figure 3: Check the concordance between the figure and its legend, as ‘C and B’ is not provided.
Figure 4: in panel C it would be better to include the response for MT/HT-C/HT (it means, heat stress with and without melatonin)
Figure 6. Move figure 6 and its description in the text from the Discussion to the Results section 3.5 (as it is an experimental result). In the Discussion, focus on new findings about effects of melatonin in both growing scenarios, control and heated.
Author Response
Dear reviewer,
Many thanks for your constructive suggestions and recognition of our research. We have made serious revisions to our manuscript.
Point 1: L 22 ‘resistance properties’ explain resistance to what (stomatic? mechanic? of membranes?).
Response 1: ‘resistance properties’ on line 22 has been changed with “heat-resistance properties” (line 36 of word file).
Point 2: L 22-23: ‘Although melatonin could not alleviate the reduction of rice yield caused by continuous high temperature’ No data on seed yield or seed weight are shown in the manuscript. As they are both very important outputs for agronomy, I encourage authors to show it (or provide a citation if those data has already been published). Otherwise, mention of seed yield should be removed from the Abstract.
Response 2: This study focused on the effect of melatonin on the fine structure of rice starch at different temperatures, so we moved the mention of seed yield from the Abstract (line 36-37 of word file).
Point 3: L 102: ‘Subsequent studies have found that melatonin is also common in higher plants’. Give more context about sites in the plant where the endogenous melatonin is synthetised (roots?, leaves? meristematic tissues?).
Response 3: More context about the sites in the plant where the endogenous melatonin is synthetised was added in the manuscript (line 125-128 of word file).
Point 4: L 107: ‘Besides, exogenous melatonin has been shown to confer plant tolerance to a variety of environmental stresses’ Give more context about what is the route of entry of exogenous melatonin to the plant, and how is it transported within the plant.
Response 4: More context about the function how exogenous melatonin can participate in abiotic stress response of plants was added in the manuscript (line 140-142 of word file).
Point 5: L 116-122: Inform the growing conditions (controlled, growth chambers) and moderate the significance of the results, avoiding to scale from individual and controlled plants to rice production in farming systems.
Response 5: The growing condition has been informed and the significance of the results have been moderated (line 143-149 of word file).
Point 6: L 127: ‘Before flowering, all rice materials were cultivated’ Give more context about number and size of pots/containers, soil/subtrate characteristics, irrigation, fertilization (nutrient solution applied throughout the cycle?), plant poulation density (plants/m2), and temperature of soil (as a proxy of root temperature). All these growing conditions have demostrated to modulate the heat stress response.
Response 6: More contexts about size of containers, soil characteristics, fertilization and plant poulation density were added in the manuscript. The temperature of soil was not tested in the study, so there is not relevant data (line 153-157 of word file).
Point 7: L 131-132: ‘The other environmental conditions in the artificial climate chamber were the same as those for the plants cultivated at normal temperature.’ Heated chambersusually modifies both, temperature and air humidity, affecting vapour pression deficit (VPD) between leaf and air. Please, include calculation of VPD for both chambers (normal and heated) from temperature and air humidity values .
Response 7: According to previous calculation methods (Kadam et al.，2015), VPD for both chambers were caculated and added in the description of chamber condition (line 159-163 of word file).
Point 8: L 133-135: ‘500 mL of 200 μmol L−1 exogenous melatonin (Sigma, USA) was used to treat rice materials planted’…’exogenous substances were sprayed three times every 5 days. ‘ It is not clear the whole melatonin dosis applied, please express itper plant or per m2. It was sprayed every 5 days during the whole grain filling period? (up to physiologycal maturity?) Also, justify the dosis and concentration used (previous data, preliminary assay?). For the Discussion section, are these application volumes feasible in extensive farming systems? Is there any obvious environmental risk of releasing unusual concentrations of melatonin into the environment?
Response 8: In this study, exogenous melatonin was sprayed only three times, at 10 daf, 15 daf and 20 daf. In order to avoid misunderstanding by readers, we redescribed the scheme of melatonin treatment， which based on the previous research results. The amount of melatonin used should not have obvious environmental risks. The relevant contents are described in the discussion part (line 392-397 of word file).
Point 9: L 137: ‘Flower marking’ Flower tagging could be better.
Response 9: “flower marking” on line137 has been replaced with “flower tagging” (line 168 of word file).
Point 10: L 152: 2.4. ‘Preparation of Seed Samples’ Describe the methodology for chalkiness determination.
Response 10: The methodology for chalkiness determination has been added in the description of ‘Preparation of Seed Samples’ (line 187-188 of word file).
Point 11: L 192-193: ‘three biological replicates’ of ‘three independent experiments’ What was the experimental unit? Individual plant? Individual pot with several plants? (detail what was the observation unit that received the treatments independently). Was the whole experiment repeated three times? Was each experiment considered as blocks? Please explain the experimental design.
Response 11: More than 3 different NPB plants grown in different bucket were sampled and used for the determination of various indexes by three technical replicates (line 228 of word file).
Point 12: L 207-209: Comparing C and MT treatments, melatonin enhances SOD and CAT activities under normal temperature. Why? Is it an indicator of oxidative stress caused by melatonin per se? Could spraying melatonin on non-stressed plants cause a seed yield penalty? Explain in the Results and expand it in the Discussion.
Response 12: Enhanced SOD and CAT activities indicated that the plant has better antioxidant capacity rather than being subjected to oxidative stress. Spraying melatonin on non-stressed plants cause a seed yield penalty, we think it is a problem of plant energy mobilization and distribution. It’s speculated that the treatment of exogenous melatonin makes the plant mobilize more energy to improve the activity of antioxidant enzyme system. The analysis of these results has been expanded in the discussion section (line 397-403 of word file).
Point 13: Figure 3: Check the concordance between the figure and its legend, as ‘C and B’ is not provided.
Response 13: The legend of Figure 3 was rewritten (line 523-525 of word file).
Point 14: Figure 4: in panel C it would be better to include the response for MT/HT-C/HT (it means, heat stress with and without melatonin)
Response 14: The response for MT/HT-C/HT was added in Figure 4C (line 613 of word file).
Point 15: Figure 6. Move figure 6 and its description in the text from the Discussion to the Results section 3.5 (as it is an experimental result). In the Discussion, focus on new findings about effects of melatonin in both growing scenarios, control and heated.
Response 15: Figure 6 corresponds to the Results section 3.5 (description of gelatinization temperature) in the original manuscript, which was mistakenly marked as Figure 5. Now we have changed the graph order and removed the graph order marked in the discussion (line 314 and 378 of word file).
Author Response File: Author Response.docx
