Droughts and Thermo-Priming Enhance Acclimation to Later Drought and Heat Stress in Maize Seedlings by Improving Leaf Physiological Activity

Round 1

Reviewer 1 Report

The original article entitled "Droughts and thermo-priming enhance adaptability to later drought and heat stress in maize seedlings by improving leaf physiological activity" might deserve publication after some revisions and suggestions as reported below:

Title:

It is OK

Abstract:

It is OK

Introduction:

It is OK

Materials and Methods:

2.1: Are they protocols already known in the literature? If not, why was it decided to use this method?

2.2.3: Indicate at which microscope magnification the samples were observed. What software was used? Was the stomatal density formula already known in the literature? In this case insert a bibliographic reference.

2.2.4-2.2.5-2.2.6-2.2.7: Briefly explain what kind of protocols were used and then refer to the bibliography. Bibliographic references alone are not enough.

2.3: Bibliographic references are missing.

A curiosity... figure 8C with which software was it made????

Discussion:

It is OK

Conclusion:

The conclusions are too synthetic. Better explain the conclusions of your work. References are missing, please cite them.

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Droughts and thermo-priming enhance adaptation to later drought and heat stress in maize seedlings by improving leaf physiological activity”(agronomy-2312886 ). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to reviewers for comments on our paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Reviewer #1:

1. 1:Are they protocols already known in the literature? If not, why was it decided to use this method?

Response: Thank you for your valuable comment. Most maize-growing regions in northwest China, including the Guanzhong Plain, may experience an increase in this sensitivity under future climate conditions. Therefore, it is imperative that attention be focused on simple and effective mitigation strategies that will help to alleviate the damage to maize seedlings caused by a combination of heat and drought stress. Many previous studies have provided promising evidence that preadaptation to abiotic stress can provide plants with a beneficial signal to identify and respond to subsequent stress as a form of stress memory. However, previous studies have mainly focused on individual heat or drought stress priming, and the priming process was only once. Heat and drought stress often occur simultaneously during the early of maize growth in Guanzhong region; Compared with a single priming process, multiple stress priming may confer plants with greater tolerance to heat and drought stress. Therefore, this study hypothesised that multiple combined heat and drought priming can improve the tolerance of maize plants to subsequent heat and drought stress compared with no priming. The purposes of the present study were: (i) to investigate the difference in pigment content, stomatal morphology, photosynthesis, antioxidant defense, cellular osmoregulation, and growth between the primed and non-primed plants; and (ii) to evaluate the capability of key physiological traits to diagnose individual and combined stresses.

2. 2.3:Indicate at which microscope magnification the samples were observed. What software was used? Was the stomatal density formula already known in the literature? In this case insert a bibliographic reference.

Response: Thank you for your valuable comment. The slides with samples was observed with a Leica optical microscope (Leica Microsystems, Germany) under ×200 (Observation of number of stomata) and 1000 (Observation of stomatal length and width) magnification. Three visual fields were chosen per leaf, and three photos were taken for each visual field. Five photos were randomly selected to measure the length and width of stomata using motic images plus 2.0 software. Stomatal density was calculated as the total numbers of stomata per area using Image J software. The stomatal density formula was already known in the literature. According to the comments of reviewer, we have cited the references. Detailed additions can be seen Lines 160-163 and Lines 166-168.

3. 2.4-2.2.5-2.2.6-2.2.7:Briefly explain what kind of protocols were used and then refer to the bibliography. Bibliographic references alone are not enough.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have added traits determination procedures in 2.2.4, 2.2.5, 2.2.6, 2.2.7 and 2.2.8. Detailed additions can be seen Lines 171-260.

4. 3:Bibliographic references are missing.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have improved “data analysis” and cited the corresponding references. Detailed additions can be seen Lines 265-273.

5. A curiosity... figure 8C with which software was it made????

Response: Thank you for your valuable comment. Figure 8C was plotted using Origin 2021 (Origin Lab Corporation, Northampton, MA, United States). A small plug-in in the Origin 2021 software, the Correlation plot, can be used to draw Figure 8C. According to the comments of reviewer, we have already made further explanation in "Data analysis". Detailed additions can be seen Lines 270-273.

6. The conclusions are too synthetic. Better explain the conclusions of your work. References are missing, please cite them.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have rewritten our conclusions to better explain our work. Specific modifications can be seen in Lines 581-600.

    We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

 

 

 

 

 

 

Best regards.

Yours sincerely,

Chen Ru

Corresponding author:

Name: Xiaotao Hu

E-mail: [email protected]

Author Response File: Author Response.docx

Reviewer 2 Report

In this study, plants were exposed to drought, heat, and a combination of them in order to develop stress tolerance to the mentioned stresses. They reported a decline in attributes related to growth and photosynthesis when the plants were subjected to heat and drought stress alone. However, the decline was more pronounced when the plants were exposed to the combined heat and drought stress. After recovery, the plants were exposed to stress again and reported an increase in the activities of the antioxidant enzyme while a decrease in oxidative stress markers in the heat and drought-primed plants than the non-primed plants. The combined priming induced more tolerance than their solo priming. Conclusively, the findings are interesting, and the paper is well-written. The author has done so many efforts to defend the results. I have no major concerns and the paper can be accepted after some minor changes.

Line 236, the “NC” should be corrected to “no stress priming + no later stress” instead of NC, no stress priming + later stress. It’s a bit confusing and sounds like non-primed plants were later exposed to stress and they performed superior.

The (Pn) (gs) and (E) should be italicized. Check through the whole MS.

Line 291. (O2-) should be changed to (O₂^-).

Good luck.

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Droughts and thermo-priming enhance adaptation to later drought and heat stress in maize seedlings by improving leaf physiological activity”(agronomy-2312886 ). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to reviewers for comments on our paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Reviewer #2:

1. Line 236, the “NC” should be corrected to “no stress priming + no later stress” instead of NC, no stress priming + later stress. It’s a bit confusing and sounds like non-primed plants were later exposed to stress and they performed superior.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have corrected to “no stress priming + no later stress” instead of NC, no stress priming + later stress. Specific modifications can be seen in Lines 315, 334, 355, 373, 394, 415.

2. The (Pn) (gs) and (E) should be italicized. Check through the whole MS.

Response: Thank you for your valuable comment. According to the comments of reviewer, Pn, gs, and E have been italicized through the whole MS. Specific modifications can be seen in Lines 179-180, 336-337, 339-340, 342-344, 350-351, 440, 492-493, and 497.

3. Line 291. (O2-) should be changed to (O₂^-).

Response: Thank you for your valuable comment. According to the comments of reviewer, O2- should be changed to O₂^-. Specific modifications can be seen in Line 369.

We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

 

 

 

 

 

 

Best regards.

Yours sincerely,

Chen Ru

Corresponding author:

Name: Xiaotao Hu

E-mail: [email protected]

Author Response File: Author Response.docx

Reviewer 3 Report

The main strength of the work is the study of a combined priming for drought and heat stress. Many biochemical and physiological parameters are investigated. Nevertheless, basic biological concepts are used incorrectly, which leads to a misinterpretation of several experimental data. Please find the main observations and advises in the followings.

- in different parts of the manuscript: in order to use the scientific terms as correctly as possible, please revise the use of the terms “adaptation” and “adaptability”: adaptation refers to an evolutionarily given set of phenotypic characteristics exhibited by every individual of a species, being inherited and stable throughout the life span of individuals, while reversible physiological changes which serve a better tolerance of adverse environmental effects are manifestations of the individual acclimation, not of the inherited adaptation; also note the relevant difference between resistance (avoidance, without changes) and tolerance (which implies genetic and metabolic changes during a process of hardening) – e. g. in row 59 instead of “improving stress resistance” it is more correct to say “improving stress tolerance”; please replace “resistance” with “tolerance” wherever it is appropriate in the manuscript

- row 51 and in several other parts of the manuscript: POD is a generic abbreviation for the sum of enzymes which catalyze peroxidation of various substrates, so POD does not stand for a specific enzyme (such as SOD or CAT), but it represents a group of different peroxidases (please use the plural)

- row 109: please explain why the temperature treatment with 36°C/28°C can be considered a “more extreme heat stress”, taking into account that for C4 type plants, such as maize, the optimal temperature range for photosynthetic carbon assimilation is appreciated to be 30-35°C; in this context, would 36°C really represent an extreme heat stress for maize?

- rows 148-149: it is not clear which one is the “paraxial” surface of the leaves; usually, the leaf surface corresponding to the upper face is called “adaxial” (closer to the axis of the corm), while the lower surface is named “abaxial” (further from the stem apex)

- rows 153-154: stomatal conductance is not identical with stomatal density, because conductance also depends on how much the stomatal pores are opened, not only on the number of stomata per leaf area

- rows 165-166: please give a brief recipe for determination of membrane injury index and malondialdehyde content (which parts of the plants were used, in what quantity, what was use for blank etc.), as it is given for the superoxide content

- rows 172-183: usually enzyme activities are expressed on a total protein content basis, for which the protein content of the plant material has to be determined; was the protein content of maize leaves used for enzyme measurements determined, and if yes, what was the method that was used for this purpose?

- rows 184-185: soluble proteins, being macromolecules, are not osmotically active, so they cannot be included in the “osmoregulatory substances”

- row 225: an increment of chlorophyll b content with 41.9% and of carotenoid content with 52.9% in plantlets exposed to heat and drought priming, as compared to unprimed control, seems very unrealistic with respect to the existing scientific literature; please verify if the relational basis is correct, e. g. the huge differences may be due to the different water content of heat- and drought-exposed and control plants, if pigment content is expressed on a fresh weight basis

- row 247: it is not clear if the increment of stomatal width with 78.5% in primed plants is due to the development of larger guard cells during leaf formation or to the curved shape of guard cells, associated with a larger pore between them

- rows 287-288: the affirmation “compared with the HDP seedlings, the HDP seedlings had a 36.77% increase in MDA content at 6 DAS” is not clear, please make the correction

- row 306: a 22.11% higher APX activity does not seem to be “remarkable”, as compared to the 54% increment of CAT activity

- rows 397-399: it is unclear what would the affirmation “the synergistic elimination of the excessive excited energy collected by the carotenoid and chlorophyll binding protein complex of the photosystem, … as a key mechanism for photosynthesis” mean; where the excessive energy originates from under the given photon flux density, and what would be the role of pigment-binding proteins in this process?

- rows 414-415: it is highly unclear how higher stomatal length can contribute to increasing leaf cooling ability

- rows 436-437: it is unclear how “reducing the damage of ROS to the cell membrane enhances the antioxidant capacity”, please rephrase or try to explain it briefly; usually, the cause-effect relation is that an improved antioxidant capacity reduces the concentration of reactive oxygen species, and as a consequence oxidative membrane damage will be restricted

- rows 442-443: please rephrase the affirmation “antioxidant enzymes may reduce superoxide production by preventing electron reductions from oxygen to superoxide”, because it seems incorrect; antioxidant enzymes do not prevent electron transfer to oxygen, they generally annihilate different forms of ROS; e. g. superoxide dismutase transforms superoxide radicals into hydrogen peroxide and oxygen

- rows 477-478: variations in chlorophyll and carotenoid content on a fresh weight basis are not relevant if variations in water content are not known, so please be circumspect when formulating conclusions based on this parameter.

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Droughts and thermo-priming enhance adaptation to later drought and heat stress in maize seedlings by improving leaf physiological activity”(agronomy-2312886 ). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to reviewers for comments on our paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Reviewer #3:

1. In different parts of the manuscript: in order to use the scientific terms as correctly as possible, please revise the use of the terms “adaptation” and “adaptability”: adaptation refers to an evolutionarily given set of phenotypic characteristics exhibited by every individual of a species, being inherited and stable throughout the life span of individuals, while reversible physiological changes which serve a better tolerance of adverse environmental effects are manifestations of the individual acclimation, not of the inherited adaptation; also note the relevant difference between resistance (avoidance, without changes) and tolerance (which implies genetic and metabolic changes during a process of hardening) – e. g. in row 59 instead of “improving stress resistance” it is more correct to say “improving stress tolerance”; please replace “resistance” with “tolerance” wherever it is appropriate in the manuscript.

Response: Thank you for your valuable comment. I have benefited greatly from your valuable advice. According to the comments of reviewer, we have revised the use of the terms “adaptation” and “adaptability” throughout the manuscript. In addition, we have replaced resistance with tolerance wherever it is appropriate in the manuscript. Specific modifications can be seen in Lines 2, 12, 27, 61, 64, 70, 568, and 578.

2. Row 51 and in several other parts of the manuscript: POD is a generic abbreviation for the sum of enzymes which catalyze peroxidation of various substrates, so POD does not stand for a specific enzyme (such as SOD or CAT), but it represents a group of different peroxidases (please use the plural)

Response: Thank you for your valuable comment. According to the comments of reviewer, we have used the plural in SOD, POD and CAT in other parts of the manuscript.

3. Row 109: please explain why the temperature treatment with 36°C/28°C can be considered a “more extreme heat stress”, taking into account that for C4 type plants, such as maize, the optimal temperature range for photosynthetic carbon assimilation is appreciated to be 30-35°C; in this context, would 36°C really represent an extreme heat stress for maize?

Response: Thank you for your valuable comment. Before conducting this experiment, we had already done a statistical analysis based on the high temperature weather during the seedling stage of summer maize in the Guanzhong Plain over the past thirty years. Meteorological data from 1990 to 2019 (http://data.cma.cn) for the major maize-producing areas in the Guanzhong Plain of China showed that the average value of extreme high temperatures during the maize seedling stage was maintained at approximately 36℃. Therefore, the maximum temperature in a high-temperature growth during the subsequent stress was set to 36℃ to simulate the potentially trend of increased temperatures in northwest China occurring during the maize seedling period. We have added a figure on the change in maximum temperature to the supplementary material (Figure. S1. Maximum temperature at the maize seedling stage in the main production areas on the Guanzhong Plain in China from 1990 to 2019). Detailed additions can be seen Lines 109-115.

4. Rows 148-149: it is not clear which one is the “paraxial” surface of the leaves; usually, the leaf surface corresponding to the upper face is called “adaxial” (closer to the axis of the corm), while the lower surface is named “abaxial” (further from the stem apex).

Response: Thank you for your valuable comment. We apologise for any confusion caused by the use of inappropriate words. We have rephrased the sentence that the colorless and transparent nail polish was applied to the adaxial surface of the median portion of leaves to collect imprint samples of stoma. Specific modifications can be seen in Lines 159-160.

5. Rows 153-154: stomatal conductance is not identical with stomatal density, because conductance also depends on how much the stomatal pores are opened, not only on the number of stomata per leaf area.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have corrected this sentence. Specific modifications can be seen in Lines 167-168.

6. Rows 165-166: please give a brief recipe for determination of membrane injury index and malondialdehyde content (which parts of the plants were used, in what quantity, what was use for blank etc.), as it is given for the superoxide content.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have supplemented the methods for the determination of MII and MDA. Detailed additions can be seen Lines 185-195.

7. Rows 172-183: usually enzyme activities are expressed on a total protein content basis, for which the protein content of the plant material has to be determined; was the protein content of maize leaves used for enzyme measurements determined, and if yes, what was the method that was used for this purpose?

Response: Thank you for your valuable comment. We have previously analysed the total protein content of maize leaves for the expression of enzyme activity. According to the comments of reviewer, we have supplemented the method for the determination of total protein content in 2.2.7. Detailed additions can be seen Lines 237-242.

8. Rows 184-185: soluble proteins, being macromolecules, are not osmotically active, so they cannot be included in the “osmoregulatory substances”

Response: Thank you for your valuable comment. According to the comments of reviewer, we have removed the soluble protein from the osmoregulatory substances.

9. Row 225: an increment of chlorophyll b content with 41.9% and of carotenoid content with 52.9% in plantlets exposed to heat and drought priming, as compared to unprimed control, seems very unrealistic with respect to the existing scientific literature; please verify if the relational basis is correct, e. g. the huge differences may be due to the different water content of heat- and drought-exposed and control plants, if pigment content is expressed on a fresh weight basis.

Response: Thank you for your valuable comment. After rechecking the data, we found that when determining the chlorophyll and carotenoid content of NHDP treatment, incorrect leaf mass was entered in the formula, resulting in deviations in the Chla, Chlb, and Car contents of NHDP treatment. We have corrected the data. In addition, in order to further eliminate the difference in leaf pigment content caused by the difference in leaf water content under high temperature and drought stress. We used the dry weight (DW) of the leaves to recalculate the Chla, Chlb and Car content of each treatment. Figures 2 and 8 (A, B and C) were plotted based on the recalculated data. Specific modifications can be seen in Lines 297-308, 310, 428, 448, 450, and 452.

10. Row 247: it is not clear if the increment of stomatal width with 78.5% in primed plants is due to the development of larger guard cells during leaf formation or to the curved shape of guard cells, associated with a larger pore between them.

Response: Thank you for your valuable comment. During the observation of stomatal morphology, we observed that the primed plants had larger guard cells than the non-primed plants, which resulted in significantly higher stomatal lengths in the primed plants than the primed plants, meanwhile, the greater stomatal width was mainly due to the greater curvature of the guard cells, which led to a larger pore between them, especially on 6 DAS. We have added corresponding explanations in the discussion section. Detailed additions can be seen Lines 500-506.

11. Rows 287-288: the affirmation “compared with the HDP seedlings, the HDP seedlings had a 36.77% increase in MDA content at 6 DAS” is not clear, please make the correction.

Response: Thank you for your valuable comment. We have corrected this sentence according to the comments of reviewer. Specific modifications can be seen in Line 366.

12. Row 306: a 22.11% higher APX activity does not seem to be “remarkable”, as compared to the 54% increment of CAT activity.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have rewritten the sentences. Specific modifications can be seen in Lines 383-387.

13. Rows 397-399: it is unclear what would the affirmation “the synergistic elimination of the excessive excited energy collected by the carotenoid and chlorophyll binding protein complex of the photosystem, … as a key mechanism for photosynthesis” mean; where the excessive energy originates from under the given photon flux density, and what would be the role of pigment-binding proteins in this process?

Response: Thank you for your valuable comment. To reduce confusion for the reader, we have removed this sentence and added the appropriate discussion and explanation. Specific modifications can be seen in Lines 477-486.

14. Rows 414-415: it is highly unclear how higher stomatal length can contribute to increasing leaf cooling ability.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have provided corresponding explanations in the Discussion. Detailed additions can be seen Lines 500-510.

15. Rows 436-437: it is unclear how “reducing the damage of ROS to the cell membrane enhances the antioxidant capacity”, please rephrase or try to explain it briefly; usually, the cause-effect relation is that an improved antioxidant capacity reduces the concentration of reactive oxygen species, and as a consequence oxidative membrane damage will be restricted.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have rephrased the phrase. Specific modifications can be seen in Lines 546-549.

16. Rows 442-443: please rephrase the affirmation “antioxidant enzymes may reduce superoxide production by preventing electron reductions from oxygen to superoxide”, because it seems incorrect; antioxidant enzymes do not prevent electron transfer to oxygen, they generally annihilate different forms of ROS; e. g. superoxide dismutase transforms superoxide radicals into hydrogen peroxide and oxygen.

Response: Thank you for your valuable comment. According to the comments of reviewer, we have rephrased the sentence. Specific modifications can be seen in Lines 530-531 and 536-543.

17. Rows 477-478: variations in chlorophyll and carotenoid content on a fresh weight basis are not relevant if variations in water content are not known, so please be circumspect when formulating conclusions based on this parameter.

Response: Thank you for your valuable comment. We have recalculated the chlorophyll and carotenoid content on a dry weight basis. Therefore, the conclusions obtained based on the results can be considered accurate.

 

We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

We look forward to your information about my revised papers and thank you for your good comments.

 

 

 

 

 

 

Best regards.

Yours sincerely,

Chen Ru

Corresponding author:

Name: Xiaotao Hu

E-mail: [email protected]

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The manuscript has been sufficiently improved to warrant publication in Agronomy. One remark for the corrected manuscript: one of my suggestions has been misunderstood: I have suggested to replace the term "adaptation" with the term "acclimation".

Author Response

Dear Editors and Reviewers:

On behalf of my co-authors, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Droughts and thermo-priming enhance adaptation to later drought and heat stress in maize seedlings by improving leaf physiological activity”(agronomy-2312886 ). We have studied reviewer’s comments carefully and have made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments which we hope meet with approval. Attached please find the revised version, which we would like to submit for your kind consideration. We would like to express our great appreciation to reviewers for comments on our paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Reviewer #3:

1. The manuscript has been sufficiently improved to warrant publication in Agronomy. One remark for the corrected manuscript: one of my suggestions has been misunderstood: I have suggested to replace the term "adaptation" with the term "acclimation".

Response: Thank you for your valuable comment. According to the comments of reviewer, we have replaced the term "adaptation" with the term "acclimation".

We have tried our best to revise and improve the manuscript according to the Reviwers′ good comments. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.docx