The Role of Nitrogen in Inducing Salt Stress Tolerance in Crocus sativus L.: Assessment Based on Plant Growth and Ions Distribution in Leaves

Round 1

Reviewer 1 Report

Hashemi et al. have provided a study about exploring the role of nitrogen in inducing salt stress tolerance in Crocus sativus plants. The topic is interesting, however, there are following crucial points that can be considered by authors to increase the value of the manuscript and may be readability. First of all,

-Title can be changed. I suggest “The role of nitrogen in inducing salinity stress tolerance in Crocus sativus L.: assessment based on plant growth and ions distribution in leaves”.

-Line 17: in inducing salt stress tolerance in plants.

-I would like to suggest to authors that the novelty and the future prospect of the study should be mentioned in the abstract and the introduction.

-Please add a paragraph providing general information on salinity stress and its mechanism in introduction and cite the studies. For example: High soil salinity affects 20–50% of irrigated agricultural land with an approximate annual economic drop of 12.6 billion USD. In most of the plant species, salinity stress can be categorized into two forms, osmotic stress and ion toxicity. As NaCl is the most prevalent salt, plants prefer its accumulation as compared to other elements available in low concentrations, such as potassium ions. Moreover, most of the plants are capable of excluding sodium and chloride ions from roots when water is absorbed from the soil. Ion toxicity occurs when over-accumulation of salt (predominantly Na+ ions) occurs in the cytoplasm of the plant cells, thereby inhibiting photosynthetic activity, protein synthesis, and other developmental processes. Where on the one hand, osmotic stress is dealt with osmotic tolerance, on the other hand, ionic stress is dealt with Na+ exclusion and tissue tolerance (Pandey et al. 2019; https://doi.org/10.3390/plants8100364).

-Please mention that which genotype of Crocus sativus was used in the study.

-Please mention one-way ANOVA in the statistical analysis.

-Table and Figure captions should be self-explanatory, please elaborate them. For example, the name of the studied plant and different treatments should be present in each caption.   

I do believe that the manuscript can be accepted once the authors address the mentioned points and enrich the manuscript with the crucial information.

Author Response

Hashemi et al. have provided a study about exploring the role of nitrogen in inducing salt stress tolerance in Crocus sativus plants. The topic is interesting, however, there are following crucial points that can be considered by authors to increase the value of the manuscript and may be readability.

Comment: -Title can be changed. I suggest “The role of nitrogen in inducing salinity stress tolerance in Crocus sativus L.: assessment based on plant growth and ions distribution in leaves”.

Response: Thanks for nice suggestion of the reviewer, we changed the title based on the reviewer`s comment as following:

The role of nitrogen in inducing salt stress tolerance in Crocus sativus L.: assessment based on plant growth and ions distribution in leaves

Comment:-Line 17: in inducing salt stress tolerance in plants.

Response: Thanks for right comment, it was corrected as following:

The role of nitrogen in inducing salt stress tolerance in plants is not well understood, and the question is more complicated in saffron (Crocus sativus L.), that is greatly sensitive to nitrogen rate.

Comment:-I would like to suggest to authors that the novelty and the future prospect of the study should be mentioned in the abstract and the introduction.

Response: Thanks the reviewer for this useful comment, we highlighted the novelties in the abstract and introduction sections, as well as present some suggestion for the future prospect in conclusion sections. The new contents follow:

The role of nitrogen in inducing salt stress tolerance in plants is not well understood, and the question is more complicated in saffron (Crocus sativus L.), that is sensitive to both nitrogen rate and salinity.

Our finding suggested that the optimum N supply (50 kg N ha^-1) strengthened the plant under non-saline and moderately saline (6 dS m^-1) conditions and consequently improved salt tolerance.

Some experimental evidences have demonstrated that saffron can be cultivated with brackish or semi-saline water, however, this feature has been neglected in the literature that contains little information about salinity tolerance of saffron is. On the other hand, there is no general consensus about the effect of N in saline conditions, it is not totally understood whether N improves salinity tolerance or intensifies the negative effect of salt stress. Therefore, our research was designed to answer these two questions: 1) what is the salt tolerance threshold of saffron? and 2) Can increasing nitrogen levels improve salinity tolerance of saffron? To fill this gap of information was the main aim of this study, where the Crocus sativus L. response to various salt stress conditions at different N supply in a controlled environment was evaluated.

Comment:-Please add a paragraph providing general information on salinity stress and its mechanism in introduction and cite the studies. For example: High soil salinity affects 20–50% of irrigated agricultural land with an approximate annual economic drop of 12.6 billion USD. In most of the plant species, salinity stress can be categorized into two forms, osmotic stress and ion toxicity. As NaCl is the most prevalent salt, plants prefer its accumulation as compared to other elements available in low concentrations, such as potassium ions. Moreover, most of the plants are capable of excluding sodium and chloride ions from roots when water is absorbed from the soil. Ion toxicity occurs when over-accumulation of salt (predominantly Na+ ions) occurs in the cytoplasm of the plant cells, thereby inhibiting photosynthetic activity, protein synthesis, and other developmental processes. Where on the one hand, osmotic stress is dealt with osmotic tolerance, on the other hand, ionic stress is dealt with Na+ exclusion and tissue tolerance (Pandey et al. 2019; https://doi.org/10.3390/plants8100364).

Response: Thanks for good suggestion, we revised the suggested content and added to the manuscript as following:

Soil salinity affects irrigated agricultural land by 20%–50% with an approximate annual economic loss more than 12.6 billion USD. The impact of salt stress on plants categorize into two phases: osmotic stress and ion toxicity. As NaCl is the most prevalent salt, plants prefer its accumulation as compared to other elements available in low concentrations, such as potassium ions. Moreover, most of the plants are capable of excluding sodium and chloride ions from roots when water is absorbed from the soil. Ion toxicity occurs when over-accumulation of salt occurs in the plant cytoplasm, thereby inhibiting photosynthetic activity, protein synthesis, and other developmental processes. While osmotic stress is dealt with osmotic tolerance, on the other hand, ionic stress is dealt with Na+ exclusion and tissue tolerance (Pandy et al., 2019).

Comment:-Please mention that which genotype of Crocus sativus was used in the study.

Response: We grateful of the reviewer, as this requested information is necessary. As the reviewer also knows, saffron propagation is done asexually and by corms, so there are no specific genotypes and cultivar for saffron as there are for other plants. This topic was added to the article as follows:

 Saffron corms are obtained through asexual reproduction, and the corms needed for this experiment were Torbat ecotype obtained from Tortbat Heydarieh as one of the most important saffron production region in the world.

Comment:-Please mention one-way ANOVA in the statistical analysis.

Response: Our statistical design was randomized complete block design (RCBD) arranged in a factorial experiment. We revised the 2.3. Statistical analyses subsection as follows:

Data in a factorial set were subjected to analysis of variance (two-way ANOVA), and the means were separated using the least significant difference (LSD) test at 5% probability level, or the standard error values (±SE). The statistical analyses were conducted by SAS software version 9.1.

Comment:-Table and Figure captions should be self-explanatory, please elaborate them. For example, the name of the studied plant and different treatments should be present in each caption.

 Response: Thanks for good comment if the reviewer, we checked all captions of Tables and Figures throughout the manuscript and add necessary details to make them self-explanatory.

Comment: I do believe that the manuscript can be accepted once the authors address the mentioned points and enrich the manuscript with the crucial information.

Thanks for your useful comments that made the manuscript better.

Author Response File: Author Response.docx

Reviewer 2 Report

I have noted major revision to catch your attention

Abstract: Please add implication of your study in the end of abstract

Introduction: Kindly provide the aim of your study in the last paragraph of Introduction.

Materials and Methods: Add how to get leaf, shoot, and corm growth in the methods

Results: Provide properties measurements Tables 1-3 with SE

Conclusions: Rewrite the conclusions answer to your aim of study. Do not repeat the results. Redo.

Author Response

I have noted major revision to catch your attention

Thank you so much for your good comments, we found all of them useful.

Comment: Abstract: Please add implication of your study in the end of abstract

Response: We grateful of the reviewer, we highlighted implication at the end of abstract as following:

Our finding suggested that the optimum N supply (50 kg N ha^-1) strengthened the plant under non-saline and moderately saline (6 dS m^-1) conditions and consequently improved salt tolerance.

Comment: Introduction: Kindly provide the aim of your study in the last paragraph of Introduction.

Response: Thanks the reviewer for this useful comment, we highlighted the aim and novelties at the end of introduction sections as follows:

The role of nitrogen in inducing salt stress tolerance in plants is not well understood, and the question is more complicated in saffron (Crocus sativus L.), that is sensitive to both nitrogen rate and salinity.

Our finding suggested that the optimum N supply (50 kg N ha^-1) strengthened the plant under non-saline and moderately saline (6 dS m^-1) conditions and consequently improved salt tolerance.

Some experimental evidences have demonstrated that saffron can be cultivated with brackish or semi-saline water, however, this feature has been neglected in the literature that contains little information about salinity tolerance of saffron is. On the other hand, there is no general consensus about the effect of N in saline conditions, it is not totally understood whether N improves salinity tolerance or intensifies the negative effect of salt stress. Therefore, our research was designed to answer these two questions: 1) what is the salt tolerance threshold of saffron? and 2) Can increasing nitrogen levels improve salinity tolerance of saffron? To fill this gap of information was the main aim of this study, where the Crocus sativus L. response to various salt stress conditions at different N supply in a controlled environment was evaluated.

Comment: Materials and Methods: Add how to get leaf, shoot, and corm growth in the methods

Response: Thank you, we described detail on measurements on leaf, shoot and corm in material and methods section as following:

Leaves length and number were measured four times during the growing seasons. The leaves length and number of the all five plants in 2018 and five randomly selected plants in 2019 were measured. Saffron plants were kept cultivating after flowering as long as leaves dried out (i.e., April 25, 2018 and April 23, 2019). At the end of the experiments of both years, the dried leaves were harvested for measuring the dry weight and ions con-centration (sodium, potassium, chlorine, calcium and magnesium). Dry weight was obtained after keeping the samples in the oven at 70±2 °C for 48 hours. The concentration of sodium (Na⁺) and potassium (K⁺) were measured with a flame photometer, concentration of chlorine (Cl^‒) was obtained with titration method and spectrophotometry was used for measuring calcium (Ca²⁺) and magnesium (Mg²⁺) concentrations. In the second year, the corms were removed from the soil and their weight and number were calculated.

Comment: Results: Provide properties measurements Tables 1-3 with SE

Response: Your potential useful suggestion is appreciated, however the information provided in Tables 1 to 3 is related to the physicochemical characteristics of soil, cow manure and water used in this experiment. All of them were analyzed once, so we cannot calculate the standard error (SE) for them because they do not repeat.

Comment: Conclusions: Rewrite the conclusions answer to your aim of study. Do not repeat the results.

Response: We grateful of the reviewer, we revised the conclusion section based on the reviewer`s comment as following:

Saffron is much more sensitive to N supply rate than other plants, because high N rate in the first season can stimulate vegetative growth and as a result, the yield is reduced or even close to zero. Sensitivity of saffron to N is more complicated in saline conditions. Salt stress had a negative effect on the saffron growth, and worsened the ions balance to the detriment of the plant. The role of Na⁺, Cl^─ and K⁺ were more important than other ions. N supply at optimum rate (50 kg ha^-1) improved the growth, however, higher rates had a negative effect. Under moderate salinity conditions (up to 6 dS m^-1), 50 kg N ha^-1 strengthened the plant and thus increased salinity tolerance, whereas, at higher salinity (i.e., 9 dS m^-1), medium and high nitrogen rates (i.e., 50 and 100 kg ha^-1) either had no effect or negative on saffron. Therefore, according to results from this research work, high amounts of N supply should be avoided in saline conditions and a supply of 50 kg N ha^-1 approximately is recommended. More researches are needed to clarify the molecular and physiological mechanisms of the nitrogen- salinity interaction, the effect of other nutrients to improve the salt tolerance of saffron, and to monitor the status of nitrogen and other elements in saline soils.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors investigated the role of nitrogen in affecting plant salinity tolerance. Their results are potentially interesting and useful. I may have the following major and minor comments.

1.     The key result from your abstract is not clear. You need to be very sharp on your key conclusions.

2.     The conclusions and implications are not much attractive. They should be built on your key results.

3.      It is unclear from your introduction that why do you want to investigate salinity tolerance? Why are they important? Why do you focus on the impacts from nitrogen? See relevant publication, Liu et al., 2021, https://doi.org/10.1016/j.soilbio.2021.108403;

4.     There is quite large room to improve the writing. Not only for the writing itself but also for the writing logic. The research questions and hypotheses are not clear. 

5.     To make your results comparable, more details on Materials and methods are required for the further evaluation. More information on the climatic, edaphic and environmental variables are required.

6.     More information on the land use history and plant and understory community composition are required. More details on the crop management are required.

7.     Relevant citations are required for your method section.

8.      Several unnecessary abbreviations are preventing the reading. I have to remember a lot abbreviations when reading your manuscript.

9.      You need more efforts for the results section to well focus on your key findings. This can make your results clearer.

10.  Some in-depth data analyses and particularly data interpretation is required to explore the underlying mechanisms. Regression analysis may help us understanding the relationships behind. Will you compare the relative importance of these variables? Have you tried to discuss the potential drivers?

11.  Tables. Decimals should be consistent across the manuscript.

12.  Some sentences are unnecessary long with changing focuses. It is not easy to understand these long sentences. The writing needs to be improved.

13.  The main conclusions and key implications are not clear enough from your discussion. The potential mechanisms need to be discussed. What are the important biotic and abiotic factors affecting the responses? What are the key implications? How can we advance the understanding of N and P cycling from this study? For example, Luo et al., 2022, https://doi.org/10.1111/1365-2435.14178

14.  Fig. 1. Missing dashed lines in other panels.

15.  Figures, it is not easy to distinguish the treatments in your figures.

16.  Figure 5, we need to report the statistical significance.

Author Response

The authors investigated the role of nitrogen in affecting plant salinity tolerance. Their results are potentially interesting and useful. I may have the following major and minor comments.

Comment: The key result from your abstract is not clear. You need to be very sharp on your key conclusions.

Response: We grateful of the reviewer, we revised the abstract section based on the reviewer`s comment as following:

The role of nitrogen (N) in inducing salt stress tolerance in plants is not well understood, and the question is more complicated in saffron (Crocus sativus L.), that is sensitive to both nitrogen rate and salinity. The present study was conducted to investigate the effects of different N (0, 50 and 150 kg ha^-1) supplies on saffron growth and ions concentration in shoot under several salt stress levels (0, 3, 6 and 9 dS m^-1). Salinity negatively affected plant growth assessed by leaves number, leaves length, shoot dry weight, corms number and corms weight. Moreover there was a clear direct correlation between higher salinity value and less plant growth. Different effects due to salinity and nitrogen were evident in terms of leaves number and length during the growing season from day 60 after first irrigation (DAF) and achieved the peak after 90 DAF. Salt stress also affected the ions balance, as Na⁺, Cl^‒ and Ca²⁺ enhanced and K⁺ reduced thus damaged the plant. Nitrogen partially mitigated the negative impacts of salinity on plant growth and ions balance, although this compensatory effect was observed when nitrogen supply was set at 50 kg N ha^-1. For example, In 2019-20, the losses in shoot dry weight due to 9 dS m^-1 salinity amounted to 47%, 44% and 54%, at 0, 50 and 100 kg N ha^-1 respectively, thus indicating a less negative effect of salinity at 50 kg N ha-1. Moreover, at 100 kg N ha^-1 the negative effect of salinity was stronger for 6 and 9 dS m^-1. Our finding suggested that the optimum N supply (50 kg N ha^-1) strengthened the plant under non-saline and moderately saline (6 dS m^-1) conditions and consequently improved salt tolerance.

Comment: The conclusions and implications are not much attractive. They should be built on your key results.

Response: Thanks the reviewer, we revised the conclusion section based on the reviewer`s comment as following:

Saffron is much more sensitive to N supply rate than other plants, because high N rate in the first season can stimulate vegetative growth and as a result, the yield is reduced or even close to zero. Sensitivity of saffron to N is more complicated in saline conditions. Salt stress had a negative effect on the saffron growth, and worsened the ions balance to the detriment of the plant. The role of Na⁺, Cl^─ and K⁺ were more important than other ions. N supply at optimum rate (50 kg ha^-1) improved the growth, however, higher rates had a negative effect. Under moderate salinity conditions (up to 6 dS m^-1), 50 kg N ha^-1 strengthened the plant and thus increased salinity tolerance, whereas, at higher salinity (i.e., 9 dS m^-1), medium and high nitrogen rates (i.e., 50 and 100 kg ha^-1) either had no effect or negative on saffron. Therefore, according to results from this research work, high amounts of N supply should be avoided in saline conditions and a supply of 50 kg N ha^-1 approximately is recommended. More researches are needed to clarify the molecular and physiological mechanisms of the nitrogen- salinity interaction, the effect of other nutrients to improve the salt tolerance of saffron, and to monitor the status of nitrogen and other elements in saline soils.

Comment: It is unclear from your introduction that why do you want to investigate salinity tolerance? Why are they important? Why do you focus on the impacts from nitrogen? See relevant publication, Liu et al., 2021, https://doi.org/10.1016/j.soilbio.2021.108403

Response: Thanks the reviewer for this useful comment, we added some content to the introduction to supply the reviewer`s comment. We also used the paper suggested by the reviewer. The new contents follow:

Soil salinity affects irrigated agricultural land by 20%–50% with an approximate annual economic loss more than 12.6 billion USD. The impact of salt stress on plants categorize into two phases: osmotic stress and ion toxicity. As NaCl is the most prevalent salt, plants prefer its accumulation as compared to other elements available in low concentrations, such as potassium ions. Moreover, most of the plants are capable of excluding sodium and chloride ions from roots when water is absorbed from the soil. Ion toxicity occurs when over-accumulation of salt occurs in the plant cytoplasm, thereby inhibiting photosynthetic activity, protein synthesis, and other developmental processes. While osmotic stress is dealt with osmotic tolerance, on the other hand, ionic stress is dealt with Na+ exclusion and tissue tolerance (Pandy et al., 2019; Liu et al., 2021).

Some experimental evidences have demonstrated that saffron can be cultivated with brackish or semi-saline water, however, this feature has been neglected in the literature that contains little information about salinity tolerance of saffron is. On the other hand, there is no general consensus about the effect of N in saline conditions, it is not totally understood whether N improves salinity tolerance or intensifies the negative effect of salt stress. Therefore, our research was designed to answer these two questions: 1) what is the salt tolerance threshold of saffron? and 2) Can increasing nitrogen levels improve salinity tolerance of saffron? To fill this gap of information was the main aim of this study, where the Crocus sativus L. response to various salt stress conditions at different N supply in a controlled environment was evaluated.

Comment: There is quite large room to improve the writing. Not only for the writing itself but also for the writing logic. The research questions and hypotheses are not clear.

Response: Thanks the reviewer for this useful comment, we checked again all parts of the manuscript in terms of enriching English. We also revised the research questions and hypotheses to make it more sense. In the following you can see the last paragraph of the introduction describing aim and hypotheses:

Some experimental evidences have demonstrated that saffron can be cultivated with brackish or semi-saline water, however, this feature has been neglected in the literature that contains little information about salinity tolerance of saffron is. On the other hand, there is no general consensus about the effect of N in saline conditions, it is not totally understood whether N improves salinity tolerance or intensifies the negative effect of salt stress. Therefore, our research was designed to answer these two questions: 1) what is the salt tolerance threshold of saffron? and 2) Can increasing nitrogen levels improve salinity tolerance of saffron? To fill this gap of information was the main aim of this study, where the Crocus sativus L. response to various salt stress conditions at different N supply in a controlled environment was evaluated.

Comment: To make your results comparable, more details on Materials and methods are required for the further evaluation. More information on the climatic, edaphic and environmental variables are required.

Response: We grateful of the reviewer, as this requested information is necessary. We added some information to the text and illustrated a Figure containing daily mean temperature, precipitation, relative humidity and mean monthly sunshine hours. Furthermore, some other information related to the physicochemical characteristics of soil, cow manure and water used in this experiment were presented in Tables 1 to 3.  In the following the new contents are inserted:

Yazd as one of the driest cities in Iran has a hot desert climate (BWh in Köppen climate classification), with a yearly precipitation amount of 49 mm and 23 days of precipitation (Figure 1). In this region, summer temperatures are very frequently above 40 °C in blazing sunshine with no humidity.

Figure 1. Long-term average (1950-2020) of climatic properties including daily mean temperature, precipitation, relative humidity and mean monthly sunshine hours in Yazd

Comment: More information on the land use history and plant and understory community composition are required. More details on the crop management are required.

Response: Great comment, but this information is necessary for a field work. Our experiment was done in 2 years in a pot system. However, more details were added on the crop management as following:

Saffron corms are obtained through asexual reproduction, and the corms needed for this experiment were Tortbat ecotype obtained from Tortbat Heydarieh as one of the most important saffron production region in the world. Five uniform corms were sown in 15 L pots on October 20, 2018, filled with soil, washed sand and cow manure with 2:1:1 weight ratio. Prior to any experiment, soil samples (Table 1) and cow manure (Table 2) were tested and the physico-chemical properties were evaluated. In the growing season of the second year, the first irrigation was performed on October 18, 2019 with the related salinity treatments.

Comment: Relevant citations are required for your method section.

Response: Thanks for useful comment, we added relevant citations wherever needed. Some of them follow:

Yazd as one of the driest cities in Iran has a hot desert climate (BWh in Köppen climate classification), with a yearly precipitation amount of 49 mm and 23 days of precipitation (Akbari et al., 2021).

Saffron corms are obtained through asexual reproduction (Kafi et al., 2006), and the corms needed for this experiment were Tortbat ecotype obtained from Tortbat Heydarieh as one of the most important saffron production region in the world.

The concentration of sodium (Na⁺) and potassium (K⁺) were measured with a flame photometer (Havre, 1961), concentration of chlorine (Cl^‒) was obtained with titration method (VDLUFA, 2012) and spectrophotometry was used for measuring calcium (Ca²⁺) and magnesium (Mg²⁺) concentrations (Lee and Campbell, 1969). In the second year, the corms were removed from the soil and their weight and number were calculated.

Comment: Several unnecessary abbreviations are preventing the reading. I have to remember a lot abbreviations when reading your manuscript.

Response: We thank reviewer, we tried our best to keep the abbreviations at the minimum number. Some of them such as nitrogen (N), sodium (Na), potassium (K), chlorine (Cl), calcium (Ca) and magnesium (Mg) are very common abbreviation for the ions.

Comment: You need more efforts for the results section to well focus on your key findings. This can make your results clearer.

Response: Thanks for this right comment, we tried our best to correct the results section and focus on our finding. These correction were done at different parts of the section.

Comment: Some in-depth data analyses and particularly data interpretation is required to explore the underlying mechanisms. Regression analysis may help us understanding the relationships behind. Will you compare the relative importance of these variables? Have you tried to discuss the potential drivers?

Response: We completely agree with the reviewer's opinion. We tried to establish the connection between our different findings in this research and brought in the results section. Also, the examination of this relationship with scientific facts and with previous reports has been discussed in the discussion section. Although it is possible to perform regression analysis, due to the different nature of the measured traits, there is no a significant and distinct trait that can be entered as a constant variable in the step-by-step regression model. Double regressions also repeat the results of Figures 2 to 6. We have made many changes in this version, which we hope will satisfy the respected reviewer.

Comment: Tables. Decimals should be consistent across the manuscript.

Response: Thank you, we checked all tables and tried to keep a same trend in decimals in all tables and values.

Comment: Some sentences are unnecessary long with changing focuses. It is not easy to understand these long sentences. The writing needs to be improved.

Response: Thank you, all parts of manuscript were checked again and necessary corrections were done for better understanding.

Comment: The main conclusions and key implications are not clear enough from your discussion. The potential mechanisms need to be discussed. What are the important biotic and abiotic factors affecting the responses? What are the key implications? How can we advance the understanding of N and P cycling from this study? For example, Luo et al., 2022, https://doi.org/10.1111/1365-2435.14178

Response: Thank you so much, based on your nice guides we revised the conclusion section. We also used the suggested paper for better discussion. New conclusion:

Saffron is much more sensitive to N supply rate than other plants, because high N rate in the first season can stimulate vegetative growth and as a result, the yield is reduced or even close to zero. Sensitivity of saffron to N is more complicated in saline conditions. Salt stress had a negative effect on the saffron growth, and worsened the ions balance to the detriment of the plant. The role of Na⁺, Cl^─ and K⁺ were more important than other ions. N supply at optimum rate (50 kg ha^-1) improved the growth, however, higher rates had a negative effect. Under moderate salinity conditions (up to 6 dS m^-1), 50 kg N ha^-1 strengthened the plant and thus increased salinity tolerance, whereas, at higher salinity (i.e., 9 dS m^-1), medium and high nitrogen rates (i.e., 50 and 100 kg ha^-1) either had no effect or negative on saffron. Therefore, according to results from this research work, high amounts of N supply should be avoided in saline conditions and a supply of 50 kg N ha^-1 approximately is recommended. More researches are needed to clarify the molecular and physiological mechanisms of the nitrogen- salinity interaction, the effect of other nutrients to improve the salt tolerance of saffron, and to monitor the status of nitrogen and other elements in saline soils.

Comment: Fig. 1. Missing dashed lines in other panels.

Response: Thank you so much for your precision, all of the Figures were redrawn emphasizing different lines. New Figures are presented after the last comment.

Comment: Figures, it is not easy to distinguish the treatments in your figures.

Response: Thank you so much for your precision, all of the Figures were redrawn emphasizing different lines for better distinguish the treatment. Furthermore, legend was added to all the Figures. New Figures are presented after the last comment.

Comment:  Figure 5, we need to report the statistical significance.

Response: Thank you so much for your precision, statistical criteria was added to the Figure 5. New Figures are presented after the last comment.

Figure 2. The effect of different nitrogen supply rates (0-100 kg N ha^-1) on leaf number of saffron (Crocus sativus L.) under varied salinities (0-9 dS m^-1) in 2018-19. The average with overlap had no significant difference based on standard error (±SE).

Figure 3. The effect of different nitrogen rates supply (0-100 kg N ha^-1) on leaf number of saffron (Crocus sativus L.) under varied salinities (0-9 dS m^-1) in 2019-20. The average with overlap had no significant difference based on standard error (±SE).

Figure 4. The effect of different nitrogen supply rates (0-100 kg N ha^-1) on leaf length of saffron (Crocus sativus L.) under varied salinities (0-9 dS m^-1) in 2018-19. The average with overlap had no significant difference based on standard error (±SE).

Figure 5. The effect of different nitrogen rates (0-100 kg N ha^-1) on leaf length of saffron (Crocus sativus L.) under varied salinities (0-9 dS m^-1) in 2019-20. The average with overlap had no significant difference based on standard error (±SE).

Figure 6. The effect of different nitrogen supply rates (0-100 kg N h^-1) on shoot dry weight in 2018-19 and 2019-20, and on corm number and weight (±SE) of saffron (Crocus sativus L.) in 2019-20 under varied salinities (0-9 dS m^-1). The average with overlap had no significant diffe

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have implemented the suggestions.

Reviewer 2 Report

The authors have addressed my comments and suggestions, I recommend to accept in the present form