Review Reports - Exogenous Spermidine Optimizes Nitrogen Metabolism and Improves Maize Yield under Drought Stress Conditions

Round 1

Reviewer 1 Report

The manuscript describes the role of spermidine in modulating nitrogen metabolism under drought in maize. Enzymes involved in nitrogen metabolism were extensively studied. A transcriptomic study and a pot trial confirming the effect on growth and yield were also given. The experiments are well designed with suitable treatments.

However, I have some doubts which need clarification.

Insert line numbers to properly indicate the comments.

Title

Not appropriate. Please re-write. Remove field verification word. Pot trial is not field verification.

Abstract

· Write the word “in hydroponics experiment” before starting the results of experiment 1.

· There is a mistake in the results written in the abstract. Check increase/decrease properly.

· Check the result of GDH. It was increased in one variety and decreased in other. Write accordingly.

· Have you measured Free amino acid? If not, why it has been written in the abstract.

Introduction

· Please check the full form of GS and GOGAT written in the whole text. Synthase/sythatase. There is an error somewhere.

Material and Methods and results

· Please mention the method of NO₃- estimation.

· Mention reference for collecting xylem fluid.

· In section 2.2 is it 500ml Chloroform in the procedure? Please verify it once again.

· In section 2.3, clearly mention plant tissue, whether it was measured from leaf or what?

· For transcriptome analysis, how many replicates were used? Why genes of some specific pathways mentioned in the manuscript were not validated by RTPCR analysis?

· After 24 hr, you are adding spermidine in PEG+spermidine treatment and after 24 hr collecting samples for transcriptomic also? Please clear the confusion.

· Whether PEG stress is for 24 hrs or for a long duration? Up to how many days the Hoagland experiment was continued?

· In PEG+spermidine treatment, spermidine was added after 24 hrs, then samples collected at 1d, should not give a significant difference in PEG+spermidine and only PEG treatment. But it is not like that. Please explain.

· Remove some repeated words in section 2.1

· What is 25L ½?

· Correct the first sentence of section 3.1

· Measurements were taken at 1d, 2d, 3d, 4d intervals. Whether the leaf samples were collected from the same seedling on each day or leaves from different seedlings were sampled on every collection day? Please clearly write it in material and methods so that there is no confusion for readers.

· Table 6 and 7 can be given as supplementary material. You should also give an overall alignment summary in an excel file in the supplementary material.

· Whether the sequencing data was submitted in any depository? Before that, it should not be published.

· In section 3.8 verify the numbers written. 83 up-regulated genes in Ck v/s Spermidine treatment? Check in each groups.

· There is some difference in points coming in the percentage increase/decrease written in the results. Please verify each figure once again.

· Correct the title of section 3.5

· Why was only Xianyu 335 variety tested in pots?

· It is already a drought resistance variety, what is the rationale behind selecting a drought resistance variety for checking drought resistance by spermidine application?

· Why did drought resistance variety give a significant decrease in yield? In CK v/s DS ?

· Write days after sowing in each case where the stage of the maize was mentioned. E.g. flowering stage (60 DAS).

· How many replicates are used per treatment in DMA, root characteristics analysis (sections 2.7, 2.8)? Please be clear in pot or plot? Do you have one plant per pot or more? Six plants selected, five plants selected means? It was per treatment or what? Please be clear.

· Please write proper cation in each table and figure, explaining replicates and standard deviation also.

· Please give reference in sections 2.7, 2.8, 2.9, 2.10 for using a particular method.

· How do you measure initial soil characteristics? Do you take samples from each pot or what? Please mention the methods clearly.

· Please clearly mention the procedure for measuring photosynthesis?

· For leaf area measurement, I think the width of a maize leaf is not uniform throughout then at what particular width was used in the calculation of leaf area and why?

· Please verify the unit of root length.

· In, section 2.11, 20 plants per plot were taken, how many per treatment? Please mention clearly.

· Please verify the data of 100-seed weight once again. Isn’t fresh weight given mistakenly? Or it is a 1000-seed weight?

· If the experiment is in pots then give yield in g per plant not in kg per ha.

Discussion

· Give reference to line 6 of the discussion for NO_3- content under drought. Rewrite the sentence from line no. 6-9 of discussion.

Please check English throughout the manuscript. There is a scope for improvement.

Author Response

Dear Reviewer 1,

Thank you for helpful and valuable comments revising manuscript. We have studied comments carefully and revised portion are marked in red in the manuscript.

Comment 0: The manuscript describes the role of spermidine in modulating nitrogen metabolism under drought in maize. Enzymes involved in nitrogen metabolism were extensively studied. A transcriptomic study and a pot trial confirming the effect on growth and yield were also given. The experiments are well designed with suitable treatments. However, I have some doubts which need clarification.

Reply 0: Thank you for your recognition of our scientific research and design. Your encouragement is the biggest driving force for our future progress, and we will continue to work hard.

Comment 1: Insert line numbers to properly indicate the comments.

Reply 1: Thank you for your serious and meticulous suggestions. We have added line numbers to the manuscript.

Title

Comment 2:Not appropriate. Please re-write. Remove field verification word. Pot trial is not field verification.

Reply 2: Yes, you are right and we have removed the “field verification” word.

Abstract

Comment 3: Write the word “in hydroponics experiment” before starting the results of experiment 1.

Reply 3: Thank you for your suggestion. According to your comments, we have revised the abstract.

Comment 4: There is a mistake in the results written in the abstract. Check increase/decrease properly.

Reply 4: Thank you for your suggestion. We have revised and improved it.

Comment 5: Check the result of GDH. It was increased in one variety and decreased in other. Write accordingly.

Reply 5: Thank you for your suggestion. We have added the feature description of GDH parameter changes of Fenghe 1.

Comment 6: Have you measured Free amino acid? If not, why it has been written in the abstract.

Reply 6: Sorry, you are right.We have deleted the expression of free amino acids.

Introduction

Comment 7: Please check the full form of GS and GOGAT written in the whole text. Synthase/sythatase. There is an error somewhere.

Reply 7: Thanks a lot and we have checked full form of GS and GOGAT written and “Synthase/sythatase”. The right expressions was as followed, “glutamine synthetase (GS), glutamate synthase (GOGAT)”.

Material and Methods and results

Comment 8: Please mention the method of NO₃- estimation.

Reply 8: Thank you for your detailed modification suggestions. We have added the method of the determination of NO₃- and two references. “[57] Jia, Q.M.; Yang, L.Y.; An, H.Y.; Dong, S.; Chang, S.H.; Zhang, C.; Liu, Y.J.; Hou, F.J. 2020. Nitrogen fertilization and planting models regulate maize productivity, nitrate and root distributions in semi-arid regions. Soil and Tillage Research, 200: 104636. https://doi.10.1016/j.still.2020.104636.”

Comment 9: Mention reference for collecting xylem fluid.

Reply 9: Thanks a lot and we have added one reference for collecting xylem fluid.“[56] Wu, Y.X.; Li, Q.; Dou, P.; Kong, F.L.; Ma, X.J.; Cheng, Q.B.; Yuan, J.C. Effect of low nitrogen stress on bleeding sap characters and root activity of maize cultivars with different low N tolerance. Journal of Plant Nutrition and Fertilizer. 2017, 23, 278–288. https://doi: 10.11674/zwyy.16103.”

Comment 10: In section 2.2 is it 500ml Chloroform in the procedure? Please verify it once again.

Reply 10: Yes, you are right and we have revised them.

Comment 11: In section 2.3, clearly mention plant tissue, whether it was measured from leaf or what?

Reply 11: Yes, you are right and we have revised them.

Comment 12: For transcriptome analysis, how many replicates were used? Why genes of some specific pathways mentioned in the manuscript were not validated by RTPCR analysis?

Reply 12: Yes, you are right and we have revised them.

Comment 13: After 24 hr, you are adding spermidine in PEG+spermidine treatment and after 24 hr collecting samples for transcriptomic also? Please clear the confusion.

Reply 13: Thank you for your suggestions. We have made modifications and improvements.

Comment14: Whether PEG stress is for 24 hrs or for a long duration? Up to how many days the Hoagland experiment was continued?

Reply 14: Thank you for your suggestions. We have made modifications and improvements.

Comment 15: In PEG+spermidine treatment, spermidine was added after 24 hrs, then samples collected at 1d, should not give a significant difference in PEG+spermidine and only PEG treatment. But it is not like that. Please explain.

Reply 15: Thank you for your suggestions. We have made modifications and improvements.

Comment 16: Remove some repeated words in section 2.1

Reply 16: Yes, you are right and we have revised them.

Comment 17: What is 25L ½?

Reply 17: 25L 1/2 Hoagland nutrient solution (pH 6.3).

Comment 18: Correct the first sentence of section 3.1

Reply 18: Yes, you are right and we have revised them.

Comment 19: Measurements were taken at 1d, 2d, 3d, 4d intervals. Whether the leaf samples were collected from the same seedling on each day or leaves from different seedlings were sampled on every collection day? Please clearly write it in material and methods so that there is no confusion for readers.

Reply 19: Yes, you are right and we have revised them.

Comment 20: Table 6 and 7 can be given as supplementary material. You should also give an overall alignment summary in an excel file in the supplementary material.

Reply 20: Thank you for your suggestions. We have moved Tables 6 and 7 into supplementary files.

Comment 21: Whether the sequencing data was submitted in any depository? Before that, it should not be published.

Reply 21: The sequencing data was not submitted in any depository.

Comment 22: In section 3.8 verify the numbers written. 83 up-regulated genes in Ck v/s Spermidine treatment? Check in each groups.

Reply 22: Yes, you are right and we have revised them.

Comment 23: Please verify each figure once again.

Reply 23: Yes, you are right and we have revised them.

Comment 24: Correct the title of section 3.5

Reply 24: Yes, you are right and we have revised them.

Comment 25: Why was only Xianyu 335 variety tested in pots?

Reply 25: The potted field experiment we did was just to verify the application effect of SPD in the field. Affected by the experimental workload, we chose only one variety as the experimental material.

Comment 26: It is already a drought resistance variety, what is the rationale behind selecting a drought resistance variety for checking drought resistance by spermidine application?

Reply 26: In preliminary experiments, the changes in the dry weight of shoots and roots of seedlings, relative water content (RWC), and the content of chlorophyll, proline and malondialdehyde (MDA) were determined to select the drought resistant and drought sensitive maize varieties from the 12 maize varieties, which commonly used in Heilongjiang Province, and screen the suitable concentrations (5%, 10%, 15%, 20%, 25% and 30%) of PEG-6000 used for simulating drought stress. We found that 5% PEG-6000 promoted the seedling growth, and especially improve the growth of the roots in some varieties and have no effect on the growth in other varieties. And the chlorophyll content increased in some varieties while have no significantly changes in other varieties. The content of proline and RWC decreased in some varieties, and the MDA content have no significantly changes in these maize seedlings. 10% PEG-6000 significantly inhibited the seedling growth, and the inhibition enhanced with the increase in the concentration of PEG-6000. With the increase in the concentration of PEG-6000, the RWC and the chlorophyll content were significantly decreased, and the content of proline and MDA were significantly increased. The magnitude of change in these physiological indicators under the treatment of PEG-6000 ranging from 10% to 20% was large, and the magnitude of change in these physiological indicators under the treatment of PEG-6000 ranging from 20% to 30% was small. Thus, 15% PEG-6000 were used to simulate drought stress to determine the effect of spermidine (Spd) on drought resistance of maize.

At the same time, we determined the drought-resistance coefficient: X=Id/Iw (Id is the value of a certain indicator under drought stress, and Iw is the value of a certain indicator under normal conditions, [Zhang et al., Analysis on Criteria for Screening Drought Tolerant Maize Hybrids. Maize science, 2007, 15(5): 65-68]), the subordination value: Xu=(X-Xmin)/(Xmax-Xmin) (Xmin and Xmax are the minimum and maximum drought-resistance coefficients of a certain indicator, respectively. [Zhang et al., Comparative Study on the Drought Resistance of Maize Hybrids. Maize science, 2012, 20(3): 29-33]), and the comprehensive drought indices (the average value of subordination value of each indicator) of the plants treated with 15%PEG to evaluate the drought resistance of these maize varieties. The results showed that the comprehensive drought indices of Xianyu 335 was the highest compared with other varieties, while Fenghe 1 had the lowest comprehensive drought indices, thus we chose Xianyu 335, as the drought resistance variety, and Fenghe 1, as the drought-sensitive variety, to study the effects of exogenous Spd on drought resistance of maize seedlings.

In order to select the appropriate Spd concentration to induce drought tolerance of maize seedlings, Spd was set at four concentrations of 0.01, 0.05, 0.1, and 1mM, and added to the nutrient solution. The treatment was repeated 3 times. CK was the maize seedlings treated with 15% PEG-6000. Samples were taken after 4 days of drought treatment. The dry and fresh weight of the plants was measured. The second leaf was used to determine the RWC, the generation rate of ·O₂^-, the content of chlorophyll, H₂O₂ and MDA, electrolyte leakage (EL), the activities of protective enzymes SOD, CAT and POD. The study found that 0.05mM and 0.1mM Spd could significantly increase the activity of SOD, CAT and POD in maize seedlings, reduce the rate of ·O₂^- production, the content of H₂O₂, MDA and Chl, and EL, thus alleviate the damage induced by drought stress on maize seedlings. Thus, the 0.05mM and 0.1mM Spd were chosen to act a further study of the role of Spd in drought resistance of maize seedlings.

Comment 27: Why did drought resistance variety give a significant decrease in yield? In CK v/s DS ?

Reply 27: CK-11816.81kg/ha, DS-8790.63 kg/ha

Comment 28: Write days after sowing in each case where the stage of the maize was mentioned. E.g. flowering stage (60 DAS).

Reply 28: Yes, you are right and we have revised them.

Comment 29: How many replicates are used per treatment in DMA, root characteristics analysis (sections 2.7, 2.8)? Please be clear in pot or plot? Do you have one plant per pot or more? Six plants selected, five plants selected means? It was per treatment or what? Please be clear.

Reply 29: Thank you for your comments. This is a common academic description in the field of maize cultivation.

Comment 30: Please write proper cation in each table and figure, explaining replicates and standard deviation also.

Reply 30: Yes, you are right and we have revised them.

Comment 31: Please give reference in sections 2.7, 2.8, 2.9, 2.10 for using a particular method.

Reply 31: We didn't add these references for the following two reasons: first, these determination methods belong to the most basic determination content in the field of plant physiology; second, because the literature of the manuscript has reached 108, the length is long, so we didn't add common references.

Comment 32: How do you measure initial soil characteristics? Do you take samples from each pot or what? Please mention the methods clearly.

Reply 32: We have measured initial soil characteristics from plot.

Comment 33: Please clearly mention the procedure for measuring photosynthesis?

Reply 33: Thank you for your comments. This is a common academic description in the field of maize cultivation.

Comment 34: For leaf area measurement, I think the width of a maize leaf is not uniform throughout then at what particular width was used in the calculation of leaf area and why?

Reply 34: We are very sorry that we failed to understand your opinion.

Comment 35: Please verify the unit of root length.

Reply 35: Thanks a lot and it should be “cm”.

Comment 36: In, section 2.11, 20 plants per plot were taken, how many per treatment? Please mention clearly.

Reply 36: Thank you for your comments. This is a common academic description in the field of maize cultivation.

Comment 37: Please verify the data of 100-seed weight once again. Isn’t fresh weight given mistakenly? Or it is a 1000-seed weight?

Reply 37: This is the weight of maize grain after drying (14% moisture).

Comment 38: If the experiment is in pots then give yield in g per plant not in kg per ha.

Reply 38: This is a conventional algorithm in the field of maize cultivation, and it is not necessary to convert it into maize yield per plant.

Discussion

Comment 39: Give reference to line 6 of the discussion for NO_3- content under drought. Rewrite the sentence from line no. 6-9 of discussion.

Reply 39: Yes, you are right and we have revised them.

Comment 40: Please check English throughout the manuscript. There is a scope for improvement.

Reply 40: Thank you for your suggestion. Indeed, as you said, there are some language problems in the manuscript. I'm very sorry for the reading difficulties caused to you. We have made further refinements to the manuscript.

Ling Dong, Lijie Li, Yao Meng, Hongliang Liu, Jing Li, Yang Yu, Chunrong Qian, Shi Wei and Wanrong Gu

29 July, 2022

Reviewer 2 Report

The manuscript by Dong et al. reported agronomic, physiological, and transcriptome analyses of maize varieties Xianyu 335 and Fenghe 1 with contrasting drought tolerance. Under spermidine and PEG treatment, experimental materials exhibited variations in some surveyed traits. Results reported here could provide guidance for the achievement of better yield performance under drought stress.

Comments

Detailed information on spermidine and PEG treatment need be added, including the reason for the selection of Spd concentration (0.1 mmol L-1 Spd), PEG concentration (15%).

Can 15% PEG reflect the difference of drought tolerance between Xianyu 335 and Fenghe 1?

How to evaluate the drought tolerance of Xianyu 335 and Fenghe 1 under hydroponic experiment?

“a.b.c.d” in Table 1 – Table 5, Figure 4-7, Table 10 need be explained in caption.

section 2.7: LS-A root analysis system. Detailed information need be provided, including instrument model, etc.

section 2.1: “Water control treatment was carried out at flowering stage (July 28)”. In China, from south to north, there are spring maize and summer maize. The specific time of maize growth and development in different regions is different, but the growth stage of maize is the same. Please delete "(July 28)" and just describe the flowering period.

section 2.4: α- Ketoglutarate. Please delete the space and change the letter K to lowercase.

Tables 6 and 7 are suggested to be moved into supplementary files.

Author Response

Dear Reviewer 2,

Thank you for helpful and valuable comments revising manuscript. We have studied comments carefully and revised portion are marked in red in the manuscript.

Comment 0: The manuscript by Dong et al. reported agronomic, physiological, and transcriptome analyses of maize varieties Xianyu 335 and Fenghe 1 with contrasting drought tolerance. Under spermidine and PEG treatment, experimental materials exhibited variations in some surveyed traits. Results reported here could provide guidance for the achievement of better yield performance under drought stress.

Reply 0: Thank you for your recognition of our scientific research and design. Your encouragement is the biggest driving force for our future progress, and we will continue to work hard.

Comment 1: Detailed information on spermidine and PEG treatment need be added, including the reason for the selection of Spd concentration (0.1 mmol L-1 Spd), PEG concentration (15%). Can 15% PEG reflect the difference of drought tolerance between Xianyu 335 and Fenghe 1? How to evaluate the drought tolerance of Xianyu 335 and Fenghe 1 under hydroponic experiment?

Reply 1: In preliminary experiments, the changes in the dry weight of shoots and roots of seedlings, relative water content (RWC), and the content of chlorophyll, proline and malondialdehyde (MDA) were determined to select the drought resistant and drought sensitive maize varieties from the 12 maize varieties, which commonly used in Heilongjiang Province, and screen the suitable concentrations (5%, 10%, 15%, 20%, 25% and 30%) of PEG-6000 used for simulating drought stress. We found that 5% PEG-6000 promoted the seedling growth, and especially improve the growth of the roots in some varieties and have no effect on the growth in other varieties. And the chlorophyll content increased in some varieties while have no significantly changes in other varieties. The content of proline and RWC decreased in some varieties, and the MDA content have no significantly changes in these maize seedlings. 10% PEG-6000 significantly inhibited the seedling growth, and the inhibition enhanced with the increase in the concentration of PEG-6000. With the increase in the concentration of PEG-6000, the RWC and the chlorophyll content were significantly decreased, and the content of proline and MDA were significantly increased. The magnitude of change in these physiological indicators under the treatment of PEG-6000 ranging from 10% to 20% was large, and the magnitude of change in these physiological indicators under the treatment of PEG-6000 ranging from 20% to 30% was small. Thus, 15% PEG-6000 were used to simulate drought stress to determine the effect of spermidine (Spd) on drought resistance of maize.

Comment 2: “a.b.c.d” in Table 1 – Table 5, Figure 4-7, Table 10 need be explained in caption.

Reply 2: Thank you for your suggestions. We have revised and improved the manuscript.

Comment 3: section 2.7: LS-A root analysis system. Detailed information need be provided, including instrument model, etc.

Reply 3: Thank you for your suggestions. We have revised this sentence in the manuscript.

Comment 4: section 2.1: “Water control treatment was carried out at flowering stage (July 28)”. In China, from south to north, there are spring maize and summer maize. The specific time of maize growth and development in different regions is different, but the growth stage of maize is the same. Please delete "(July 28)" and just describe the flowering period.

Reply 4: Yes, you are right and we have deleted these words.

Comment 5: section 2.4: α- Ketoglutarate. Please delete the space and change the letter K to lowercase.

Reply 5: Thank you for your suggestions. We have uniformly revised and improved similar problems in the manuscript.

Comment 6: Tables 6 and 7 are suggested to be moved into supplementary files.

Reply 6: Thank you for your suggestions. We have moved Tables 6 and 7 into supplementary files.

We are very grateful for you raising this important question. After receiving your helpful comments yesterday, our scientific research and academic team held an emergency video conference with Tencent conference software immediately. We have studied your valuable comments carefully and we hope our reply could meet with approval. If you have any other questions, please feel free to contact us.

Best regards.

Ling Dong, Lijie Li, Yao Meng, Hongliang Liu, Jing Li, Yang Yu, Chunrong Qian, Shi Wei and Wanrong Gu

29 July, 2022

Round 2

Reviewer 1 Report

Please indicate the line numbers in your reply against each comment so that I can track your modifications easily. Sometimes it seems that you have written in reply that “Yes, you are right and we have revised them” but actually you have not revised it. I failed to track the answers to the following comments.