γ-Aminobutyric Acid Alleviates Salinity-Induced Impairments in Rice Plants by Improving Photosynthesis and Upregulating Osmoprotectants and Antioxidants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editors and Authors, I read with interest the manuscript entitled “γ-aminobutyric acid (GABA) alleviates salinity induced impairments in rice plants by improving photosynthesis and upregulating osmoprotectants and antioxidants”. In this study, GABA was applied exogenously at varying levels and evaluated its effects on yield and yield components, chloroplast and stomatal ultrastructure, antioxidant enzyme activities, chlorophyll content, and other biochemical parameters. Particular emphasis was placed on photosynthesis to investigate the physiological and biochemical mechanisms through which GABA enhances salt tolerance in rice. Therefore, the manuscript needs some adjustments so that it can then be forwarded to the publication process. The manuscript has the potential for publication in the journal Agronomy and needs the following adjustments:

TITLE

- Do not mention the abbreviation “GABA” in the title. To remove. Mention for the first time in the summary.

 

ABSTRACT

- What were the photosynthetic characteristics? Gas exchange? Fluorescence? This should be mentioned as well, like the other variables.

- What treatments were used? This should be described here. Salinity and GABA.

- Replace keywords that are repeated in the title.

- Do not use the word GABA (abbreviation) in the words -key.

 

INTRODUCTION

 

- The last sentence of the Introduction needs to be referenced. Line 44.

 

- The excerpt from line 52-56 needs to be referenced. This is very important information that needs to be cited.

 

- Line 60: Liu et al. is mentioned. and the year is not entered. Enter.

- Line 74 it was mentioned that the role of GABA is not fully understood in plants. Is this true? Are there still gaps in knowledge about this?

- Line 82 it was mentioned that GABA is crucial for several factors abiotic. These factors were mentioned in the previous paragraph, making this repetitive information. I suggest removing or replacing it. - Line 112. It is not necessary to state that the main emphasis of the study was on photosynthesis. I suggest deleting this information.

- Add some hypotheses about the research. Insert them before the objectives.

 

MATERIAL AND METHODS

 

- Climate data was collected during the experiment in the years 2020 and 2021? I suggest including a figure with the climate data.

- Were the seeds sown in trays? Mention it.

- Why were mM or dS m-1 not used for salinity? These are more common measurements when working with salinity.

- Was the sea salt NaCl?

- Was any adjuvant used in the application of GABA in the solution to improve fixation on the plants?

- How was the spraying done? Was a manual sprayer used?

- What methodology was used to quantify the photosynthetic pigments (chlorophyll a and b)? This was not mentioned. - In the gas exchange analyses, were stomatal conductance and internal CO2 concentration not measured? Why?

- What is the purpose of measuring chlorophyll and the SPAD index? The SPAD index is an estimate of total chlorophyll. I don't see any point in measuring the two things. Review.

- Line 165: It is not necessary to inform that CO2 cylinders were used.

- Line 221: Correlation of what? Mention.

- Cite the R program reference.

 

RESULTS

 

- In line 3.5: Topic 3.5: There was no comparison between the harvests (2020 and 2021). A general comparison was made between the treatments. Which test was used? This information should be presented in the caption or footer of all figures and tables.

- Table 3 presents the results of chlorophyll and SPAD . I don't see the need to use SPAD, since chlorophyll was made. DISCUSSION

 

- Add more information in topic 4.1.

 

- Review some excerpts that are not related to the main results found in this study.

 

CONCLUSION

 

- Reduce this section. Too broad.

 

- It is not necessary to re-emphasize results from previous studies. Authors should focus on the main results and novelties found in the present study.

Author Response

Title

Do not mention the abbreviation “GABA” in the title. To remove. Mention for the first time in the summary.

Response: Thanks. I have removed the abbreviation “GABA” in the title.

 

Abstract

What were the photosynthetic characteristics? Gas exchange? Fluorescence? This should be mentioned as well, like the other variables.

Response: Thanks for your mention. I have included the measurements taken for photosynthetic characteristics and revised the sentence as “We measured the activities of superoxide dismutase, peroxidase, and catalase, as well as photosynthetic characteristics such as photosynthesis, transpiration, chlorophyll content, stomatal density and size, and leaf anatomical features.”

 

What treatments were used? This should be described here. Salinity and GABA.

Response: I appreciate your suggestion. I have described the treatments in the abstract by adding “Therefore, RNA-seq (RNA sequencing) was used to analyze the differential expression of the transcriptome between CK and salinity treatments, revealing the key roles of GABA in salt tolerance. Subsequently, different levels of exogenous GABA were applied to salt-stressed plants to investigate its physiological role in enhancing salt tolerance.”

 

Replace keywords that are repeated in the title.

Response: Thanks for your suggestion. We have revised the keywords to avoid repetition. The keyword was changed to “antioxidant defense, mesophyll, osmotic regulation, RNA-seq, stomata, salt stress”

 

Do not use the word GABA (abbreviation) in the words -key.

Response: Thanks. I have removed the word GABA (abbreviation) from the keywords.

 

Introduction

The last sentence of the Introduction needs to be referenced. Line 44.

Response: Thanks. I have included a citation (Mukhopadhyay et al., 2021) regarding soil salinity mitigation strategies for sustainable agriculture and food security, which highlights planting salt-tolerant rice as an effective solution.

Reference: Mukhopadhyay, R.; Sarkar, B.; Jat, H. S.; Sharma, P. C.; Bolan, N. S. Soil salinity under climate change: Challenges for sustainable agriculture and food security. J. Environ. Manage. 2021, 280, 111736.

 

The excerpt from line 52-56 needs to be referenced. This is very important information that needs to be cited.

Response: Thanks for your suggestion. I have added references line 52-56 ( Reddy et al. 2017; Qin et al. 2020).

References:

Reddy, I.N.B.L.; Kim, B.K.; Yoon, I.S.; Kim, K.H.; Kwon, T.R. Salt tolerance in rice: focus on mechanisms and approaches. Rice Sci. 2017, 24, 123-144.

Qin, H., Li, Y., & Huang, R. Advances and challenges in the breeding of salt-tolerant rice. Int. J. Mol. Sci. 2020, 21, 8385.;

 

Line 60: Liu et al. is mentioned. and the year is not entered. Enter.

Response: I appreciate your mention. I have added the year as “Liu et al. (2023)”.

 

Line 74 it was mentioned that the role of GABA is not fully understood in plants. Is this true? Are there still gaps in knowledge about this?

Response: Thanks for the suggestion. We cite the paper of Hasan et al. (2021) and revised the sentence to “However, the exact biological role of GABA in plants under stressed environment is still not fully understood, for example, the role of GABA in the movement of stomata”.

Reference: Hasan, M.M.; Alabdallah, N.M.; Alharbi, B.M.; Waseem, M.; Yao, G.; Liu, X.D.; Abd El-Gawad, H.G.; El-Yazied, A.A.; Ibrahim, M.F.; Jahan, M.S.; Fang, X.W. GABA: A key player in drought stress resistance in plants. Int. J. Mol. Sci. 2021, 22, p.10136.

 

Line 82 it was mentioned that GABA is crucial for several factors abiotic. These factors were mentioned in the previous paragraph, making this repetitive information. I suggest removing or replacing it.

Response: Thanks for your suggestion. I have removed it.

 

Line 112. It is not necessary to state that the main emphasis of the study was on photosynthesis. I suggest deleting this information.

Response: Thanks for your mention. I have deleted this information.

 

Add some hypotheses about the research. Insert them before the objectives.

Response: Thanks. I have added a hypothesis “We hypothesized that variations in transcriptome expression patterns could help uncover the mechanisms behind the differing salt tolerance between genotypes.”

 

Material and methods

Climate data was collected during the experiment in the years 2020 and 2021? I suggest including a figure with the climate data.

Response: Thanks for your suggestion. In this manuscript, we have already included eight Figures and Five Tables. Climate data is generally necessary for agronomic studies. However, in this study we mainly focus on the physiological roles of GABA under salt stress. Therefore, we didn’t include the weather data in this manuscript.

 

Were the seeds sown in trays? Mention it.

Response: Thanks. We have revised the part as “Seeds were sown on May 12 on seedbed and transplanted into pots on June 12, with three hills per pot and two seedlings per hill.”

 

Why were mM or dS m^-1 not used for salinity? These are more common measurements when working with salinity.

Response: Thanks for your suggestion. I have added Table 1 of electrical conductivity of different treatments at different growth stages.

 

Was the sea salt NaCl?

Response: Thanks. The pots were filled with 13 kg of sieved soil per pot. Before transplanting, artificial sea salt (Blue Starfish Salt Product Co., Ltd., Zhejiang, China, 94.5% NaCl, 0.11% K⁺, 0.13% Mg²⁺, 0.06% Ca²⁺ and 3.7% SO₄^2-) was added 2.0 g kg^-1 to each pot.

 

Was any adjuvant used in the application of GABA in the solution to improve fixation on the plants?

How was the spraying done? Was a manual sprayer used?

Response: Thank you for your suggestions. We used Tween-20 as a surfactant, and the solution was applied manually by spraying. We revised the sentences as “All the spray solutions contained Tween-20 of a final concentration of 0.02% (v/v) as a surfactant. GABA was applied to both sides of the leaves during the pre-tillering and jointing stages manually.”

 

What methodology was used to quantify the photosynthetic pigments (chlorophyll a and b)? This was not mentioned. - In the gas exchange analyses, were stomatal conductance and internal CO2 concentration not measured? Why?

Response: Thanks for your mentions. We have added the methodology of measuring chlorophyll a and chlorophyll b as “At flowering stage, chlorophyll a (Chl a) and chlorophyll b (Chl b) concentrations of flag leaf were determined following the method of Jin et al. [32].”. In the gas exchange analyses, we also measured stomatal conductance and internal CO2 concentration. However, since we have already presented the results for net photosynthesis and leaf transpiration rate, it is unnecessary to include these additional measurements.

Reference: Jin, X.; Liu, T.; Xu, J.; Gao, Z.; Hu, X. Exogenous GABA enhances muskmelon tolerance to salinity-alkalinity stress by regulating redox balance and chlorophyll biosynthesis. BMC Plant Biol. 2019, 19, 1-15.

 

What is the purpose of measuring chlorophyll and the SPAD index? The SPAD index is an estimate of total chlorophyll. I don't see any point in measuring the two things. Review.

Response: Thank you for your question. We used SPAD measurements to estimate total chlorophyll content, but we could not specifically determine the amounts of chlorophyll a or chlorophyll b. This is why we also performed chlorophyll measurements.

 

Line 165: It is not necessary to inform that CO2 cylinders were used.

Response: Thanks. I have removed the CO₂ cylinders in line 165.

 

Line 221: Correlation of what? Mention.

Response: Thanks. We revised this part as “Correlation analysis of measured physiological traits was also done in SAS.”

 

Cite the R program reference.

Response: We cited the R program as “Structural equation modeling was conducted using the ‘lavaan’ package in R 4.0.2 (https://cran.r-project.org/web/packages/lavaan/index.html).”

 

Results

In line 3.5: Topic 3.5: There was no comparison between the harvests (2020 and 2021). A general comparison was made between the treatments. Which test was used? This information should be presented in the caption or footer of all figures and tables.

Response: Thanks for the suggestion. we have added in footer of all figures and tables, that Tukey’s test was used at P<0.05.

 

Table 3 presents the results of chlorophyll and SPAD. I don't see the need to use SPAD, since chlorophyll was made.

Response: Thanks. We used SPAD measurements to estimate total chlorophyll content, but we could not specifically determine the amounts of chlorophyll a or chlorophyll b. This is why we also performed chlorophyll measurements.

 

Discussion 

Add more information in topic 4.1.

Response: Thanks for your suggestion. I have revised the content of this section as: “GABA is irreversibly synthesized by glutamate decarboxylase (GAD). GAD is vital for GABA metabolism in response to abiotic stress, catalyzing both the degradation of gluta-mate (Glu) and the synthesis of GABA [39]. In our study, we analyzed the relative expression levels of GADs in the leaves of both rice genotypes under salt stress. We identified a gene (LOC4333932) in the salt-tolerant rice cultivar that increases GABA content by elevating GAD activity (Figure 2).”

 

Review some excerpts that are not related to the main results found in this study.

Response: We have deleted some parts which are not related to the main results of this study.

 

Conclusion

Reduce this section. Too broad. It is not necessary to re-emphasize results from previous studies. Authors should focus on the main results and novelties found in the present study.

Response: We have removed some redundant sentences in this section, and revised this part to “RNA-seq analysis revealed that GABA production was significantly enhanced through the activation of the glutamate decarboxylase (GAD) gene (LOC4333932) in salt-tolerant rice cultivars. The apply of GABA improves the salt tolerance of salt-tolerant rice cultivar and salt-susceptible rice cultivar by stimulating the synthesis of endogenous GABA and ABA. Under salt stress, the photosynthesis was improved and the transpira-tion was decreased by increasing of endogenous GABA and ABA after spraying exoge-nous GABA. The activity of CAT, POD and SOD and the content of Fru and Pro were in-creased by increasing of photosynthesis and the Na+/K+ was decreased by decreasing of transpiration. Therefore, Exogenous GABA application improved the salt tolerance and yield of different rice cultivars under salt stress. Moreover, analysis of stomatal density, size, and leaf anatomical characteristics showed a reduction in SPI, which lowered the transpiration rate. Concurrently, the increased proportion of mesophyll cell periphery covered by chloroplasts (Sc/Sm) indicated improved mesophyll conductance. This im-provement in internal CO₂ diffusion, along with reduced water loss, contributed to the maintenance of photosynthesis under saline environments, further enhancing rice salt tolerance. In conclusion, our study underscores the potential of exogenous GABA applica-tion as a promising strategy for mitigating salinity stress in rice production by promoting physiological adaptations that improve water-use efficiency, photosynthesis, and overall growth under adverse conditions. This approach could be crucial in sustaining crop productivity on saline soils, which are becoming increasingly prevalent due to environ-mental challenges.”

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled, "γ-aminobutyric acid (GABA) alleviates salinity induced impairments in rice plants by improving photosynthesis and up-regulating osmoprotectants and antioxidants" is not a clear study. In materials and methods, authors have mentioned two levels of salinity (0 and 2%), but in all figures and tables, I cannot see anything related to normal and saline conditions. Add the data on rice under control conditions in comparison with saline conditions. However, there are some comments which can improve this article.

 

Abstract

·         Mention the treatments

·         “In this study, we investigated the expression levels of genes related to GABA synthesis, as well as the contents of malondialdehyde, proline, fructose, Na+ , K+ and endogenous abscisic acid and GABA. We also measured the activities of superoxide dismutase, peroxidase, and catalase, along with photosynthetic characteristics”. Not need to write such lines.

·         Results were poorly explained, need to revise them and write in a mechanistic way.

·         of boxes? What kind of material have you added in these boxes? Sand, soil etc?

Introduction

·         Before this line “Rice is sensitive to salinity stress, especially during its early growth stages, which 46 can lead to substantial reductions in plant height, tillering, and grain yield” add a line about rice threshold to salinity?

·         Explain, Why is it increasing every year ? Overall, in the world the agriculture loss due to salinity in dollars.

·         Write the rice production and area in the World and in China

·         Overall, try to cite new studies and remove old.

Materials and Methods

·         salt stress (0.2%) treatments? Why have you chosen this level? And write this in mM or dS m^-1.

·         EC can not be zero, if you have normal soil, there should be some EC of that soil, mention the EC as well as a control.

·         When have you applied the salinity? How you have imposed it?

·         Why have you chosen these stage for foliar spray? “pre-tillering and jointing stages”

·         What is the dimension of pots?

·          

Results

·         In materials and methods, authors have mentioned two levels of salinity (0 and 2%), but in all figures and tables, I cannot see anything related to normal and saline conditions

 

Comments on the Quality of English Language

Minor changes are required

Author Response

Abstract

Mention the treatments

“In this study, we investigated the expression levels of genes related to GABA synthesis, as well as the contents of malondialdehyde, proline, fructose, Na+, K+ and endogenous abscisic acid and GABA. We also measured the activities of superoxide dismutase, peroxidase, and catalase, along with photosynthetic characteristics”. Not need to write such lines.

Response: Thanks for the suggestion. We have mentioned the treatment and revised this part as “Therefore, RNA-seq (RNA sequencing) was used to analyze the differential expression of the transcrip-tome between CK and salinity treatments, revealing the key roles of GABA in salt tolerance. Subsequently, different levels of exogenous GABA were applied to salt-stressed plants to investigate its physiological role in enhancing salt tolerance.”

 

 

Results were poorly explained, need to revise them and write in a mechanistic way.

Response: Thanks. I have revised the results and write in a mechanistic way as “Exogenous GABA application improves salt stress tolerance by increasing endogenous ABA and GABA contents, which enhance osmotic adjustment, boost antioxidant defenses, and regulate ion balance. These combined effects help maintain photosynthetic efficiency and support overall plant growth under salt stressed environment. Additionally, the increased proportion of mesophyll cell periphery covered by chloroplasts (Sc/Sm) indicated enhanced mesophyll conductance. These helped maintain photosynthesis under saline conditions while reducing water consumption.”

 

of boxes? What kind of material have you added in these boxes? Sand, soil etc?

Response: Thanks. The pots were filled with 13 kg of sieved soil per pot. Before transplanting, artificial sea salt (Blue Starfish Salt Product Co., Ltd., Zhejiang, China, 94.5% NaCl, 0.11% K⁺, 0.13% Mg²⁺, 0.06% Ca²⁺ and 3.7% SO₄^2-) was added 2.0 g·kg⁻¹ to each pot.

 

Introduction

Before this line “Rice is sensitive to salinity stress, especially during its early growth stages, which 46 can lead to substantial reductions in plant height, tillering, and grain yield” add a line about rice threshold to salinity?

Response: Thanks. I have added rice threshold to salinity in the manuscript as “The soil salinity levels above the critical threshold (3.0 dS m^-1) (Zheng et al. 2023) can resulted in substantial reductions in plant height, tillering, and grain yield.”

Reference: Zheng, C.; Liu, C.; Liu, L.; Tan, Y.; Sheng, X.; Yu, D.; Sun, Z.; Sun, X.; Chen, J.; Yuan, C.; Duan, M. Effect of salinity stress on rice yield and grain quality: A meta-analysis. Eur. J. Agron. 2023, 144, 126765.

 

Explain, Why is it increasing every year? Overall, in the world the agriculture loss due to salinity in dollars. Write the rice production and area in the World and in China

Response: Thanks for the suggestion. Soil salinization is increasing annually due to poor irrigation practices, rising temperatures, sea level rise, and land degradation. Over-irrigation, especially in arid regions, leaves salts behind as water evaporates, while climate change accelerates evaporation and reduces rainfall needed to flush salts from the soil. Coastal areas face saltwater intrusion from rising sea levels, and deforestation disrupts water balance, further promoting salt accumulation. Additionally, poor drainage systems prevent proper salt removal, contributing to the expansion of salinized farmland each year.

 

Overall, try to cite new studies and remove old.

Response: Thanks. I have cited new studies in the introduction.

 

Materials and Methods

salt stress (0.2%) treatments? Why have you chosen this level? And write this in mM or dS m^-1.

Response: Thanks. In our previous study (Yan et al., 2022), we found that a 0.2% salt stress treatment was optimal for observing differences between salt-susceptible and salt-tolerant rice cultivars. I have added Table 1 of electrical conductivity of different treatments at different growth stages.

Reference: Yan, J.; Gu, Y.; Xue, Z.; Zhou, T.; Ge, Q.; Zhang, H.; Liu, L.; Wang, Z.; Gu, J.; Yang, J.; Zhou, Z.; Xu, D. Different responses of rice cultivars to salt stress and the underlying mechanisms. Acta Agronomica Sinica. 2022, 48, 1463-1475.

 

 

EC can not be zero, if you have normal soil, there should be some EC of that soil, mention the EC as well as a control.

Response: I have added Table 1 of electrical conductivity of different treatments at different growth stages. In CK treatment, the electrical conductivity is about 225 μS cm^-1

 

When have you applied the salinity? How you have imposed it?

Response: Thanks. The pots were filled with 13 kg of sieved soil per pot. Before transplanting, artificial sea salt (Blue Starfish Salt Product Co., Ltd., Zhejiang, China, 94.5% NaCl, 0.11% K⁺, 0.13% Mg²⁺, 0.06% Ca²⁺ and 3.7% SO₄^2-) was added 2.0 g·kg⁻¹ to each pot.

 

Why have you chosen these stages for foliar spray? “pre-tillering and jointing stages”

Response: Thanks for suggestion. Because rice is sensitive to salinity stress, especially during its early growth stages.

 

What is the dimension of pots?

Response: Thanks. The pot has a diameter of 20 cm and a height of 35 cm.

    

Results

In materials and methods, authors have mentioned two levels of salinity (0 and 2%), but in all figures and tables, I cannot see anything related to normal and saline conditions

Response: Thanks. There were two experiments. In experiment I, RNA-seq (RNA sequencing) was used to analyze the differential expression of the transcriptome between CK and salinity treatments; In experiment II, different levels of exogenous GABA were applied to salt-stressed plants to investigate its physiological role in enhancing salt tolerance.

Reviewer 3 Report

Comments and Suggestions for Authors

The relevance of this work is high, as the authors have described in detail in the introduction. The work has been completed at a high methodological level and contains new scientific results from two summer studies on the role of GABA in salt tolerance of rice plants. The figures are high-quality and detailed, the tables are informative, and the references to literary sources are new - for the last 10 years.

There are some minor comments and suggestions for improving the work.

The Latin name of the rice species should be indicated in the abstract, keywords, and 2.1 Rice cultivation and experimental design. In addition, the rice varieties should be indicated in the abstract and 2.1 Rice cultivation and experimental design.

L. 106. The authors write that “two rice cultivars: the salt-tolerant Lianjian 5 (J-5) and the salt-susceptible Lianjing 7 (L-7).” How is this known? A reference to the source is needed.

L. 124. Was salt added to the soil only once, before planting? It is not clear from the methods.

L. 130. Or was salt also added to the soil before each GABA spraying, i.e. 4 times in total? What was the total amount of salt added to the soil?

L. 152. What method was used to determine the chlorophyll content? What do the SPAD values ​​mean?

L. 160. The measured parameters should be indicated, for example, “Pn, photosynthetic rate; Tr, transpiration rate.”

L. 200-211. The essence of each research method should be briefly described. This is due to the fact that, for example, L. 207. Reference [35] does not contain a method for determining fructose.

L. 209-211. Reference [26] does not contain detailed methods for determining antioxidant enzymes; the authors also refer to other sources. Therefore, it is not clear, for example, which form of peroxidase the authors determined.

L. 219. What do the data in the tables represent - mean values ​​and errors of the mean or standard deviation? What is the biological and analytical repeatability?

L. 289. Here and in the titles of other tables, replace “of salt-tolerant and salt-susceptible rice cultivars” with “of salt-tolerant (J-5) and salt-susceptible (L-7) rice cultivars”.

According to the data in Table 2, rice cultivars differed in tissue dry matter content. Therefore, all calculations in Table 3 should be performed on g DW, then the values ​​will be more correct. Do you agree?

L. 407 and Figure 7. The authors write “…on the contents of K+, Na+, osmotic adjustment substances, and the activities of antioxidant enzymes…”. Do the authors classify MDA, malondialdehyde as osmotic adjustment substances?

Author Response

The Latin name of the rice species should be indicated in the abstract, keywords, and 2.1 Rice cultivation and experimental design. In addition, the rice varieties should be indicated in the abstract and 2.1 Rice cultivation and experimental design.

Response: Thanks. We have added the Latin name of the rice species in the abstract, keywords and rice cultivation and experimental design. Also, we have added the rice varieties in the abstract and rice cultivation and experimental design.

 

L.106. The authors write that “two rice cultivars: the salt-tolerant Lianjian 5 (J-5) and the salt-susceptible Lianjing 7 (L-7).” How is this known? A reference to the source is needed.

Response: we selected salt-tolerant Lianjian 5 (J-5) and the salt-susceptible Lianjing 7 (L-7) based on our previous studies of Yan et al. (2022) and Li et al. (2023).

References:

Yan, J.; Gu, Y.; Xue, Z.; Zhou, T.; Ge, Q.; Zhang, H.; Liu, L.; Wang, Z.; Gu, J.; Yang, J.; Zhou, Z.; Xu, D. Different responses of rice cultivars to salt stress and the underlying mechanisms. Acta Agronomica Sinica. 2022, 48, 1463-1475

Li, Z.; Zhou, T.; Zhu, K.; Wang, W.; Zhang, W.; Zhang, H.; Yang, J. Effects of salt stress on grain yield and quality parameters in rice cultivars with differing salt tolerance. Plants. 2023, 12, 3243.

 

L. 124. Was salt added to the soil only once, before planting? It is not clear from the methods. L. 130. Or was salt also added to the soil before each GABA spraying, i.e. 4 times in total? What was the total amount of salt added to the soil?

Response: Thanks. The salt added to the soil only once. The pots were filled with 13 kg of sieved soil per pot. Before transplanting, artificial sea salt (Blue Starfish Salt Product Co., Ltd., Zhejiang, China, 94.5% NaCl, 0.11% K⁺, 0.13%Mg²⁺, 0.06% Ca²⁺ and 3.7% SO₄^2-) was added 2.0 g·kg⁻¹ to each pot.

 

L. 152 What method was used to determine the chlorophyll content? What do the SPAD values ​​mean?

Response: Thanks. At flowering stage, chlorophyll a (Chl a) and chlorophyll b (Chl b) concentrations of flag leaf were determined following the method of Jin et al. (2019). The portable chlorophyll (Chl) meter SPAD-502 Plus (Konica Minolta Optics, Japan) was used to measure leaf SPAD value.

Reference: Jin, X.; Liu, T.; Xu, J.; Gao, Z.; Hu, X. Exogenous GABA enhances muskmelon tolerance to salinity-alkalinity stress by regulating redox balance and chlorophyll biosynthesis. BMC Plant Biol. 2019, 19, 1-15.

 

L. 160. The measured parameters should be indicated, for example, “Pn, photosynthetic rate; Tr, transpiration rate.”

Response: Thanks for your suggestion. I have indicated the measured parameters in the manuscript.

 

L. 200-211. The essence of each research method should be briefly described. This is due to the fact that, for example, L. 207. Reference [35] does not contain a method for determining fructose.

Response: Thanks. I have referred to Li et al. (2020) to introducing a method for determining fructose.

Li, Z.; Qian, Z.; Cheng, F. Sugar starvation enhances leaf senescence and genes involved in sugar signaling pathways regulate early leaf senescence in mutant rice. Rice Sci. 2020, 27, 201-214.

 

L. 209-211. Reference [26] does not contain detailed methods for determining antioxidant enzymes; the authors also refer to other sources. Therefore, it is not clear, for example, which form of peroxidase the authors determined.

Response: Thanks. I have referred to another article (Gu et al. 2020) to provide a more detailed methods for determining antioxidant enzymes.

Reference: Gu, Y.; Liang, C. Responses of antioxidative enzymes and gene expression in Oryza sativa L and Cucumis sativus L seedlings to microcystins stress. Ecotox. Environ. Safe. 2020, 193, 110351

 

L. 219. What do the data in the tables represent - mean values and errors of the mean or standard deviation? What is the biological and analytical repeatability?

Response: Thanks for the suggestion. For example, in table 2, we have added “Values ± SD (n=3) in the same column of the same cultivar with different letters are significantly different between treatments (P<0.05, Tukey's test).”

 

M. 289. Here and in the titles of other tables, replace “of salt-tolerant and salt-susceptible rice cultivars” with “of salt-tolerant (J-5) and salt-susceptible (L-7) rice cultivars”.

Response: Thanks. I have replaced “of salt-tolerant and salt-susceptible rice cultivars” with “of salt-tolerant (J-5) and salt-susceptible (L-7) rice cultivars”

 

According to the data in Table 2, rice cultivars differed in tissue dry matter content. Therefore, all calculations in Table 3 should be performed on g DW, then the values will be more correct. Do you agree?

Response: Thanks for the comments. Generally, for chlorophyll measurement, most authors use the unit of per fresh weight.

 

L. 407 and Figure 7. The authors write “…on the contents of K+, Na+, osmotic adjustment substances, and the activities of antioxidant enzymes…”. Do the authors classify MDA, malondialdehyde as osmotic adjustment substances

Response: Thanks. I have already included MDA in the L. 407 and in caption of Figure 7.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors modified the manuscript according to the suggestions.

Author Response

The authors modified the manuscript according to the suggestions.

Response: Thanks for all your inputs in the reviewing.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled, "γ-aminobutyric acid (GABA) alleviates salinity induced impairments in rice plants by improving photosynthesis and up-regulating osmoprotectants and antioxidants" is not a clear study. In materials and methods, authors have mentioned two levels of salinity (0 and 2%), but in all figures and tables, I cannot see anything related to normal and saline conditions. Add the data on rice under control conditions in comparison with saline conditions. However, there are some comments which can improve this article.

Comments on the Quality of English Language

Minor changes required

Author Response

The manuscript entitled, "γ-aminobutyric acid (GABA) alleviates salinity induced impairments in rice plants by improving photosynthesis and up-regulating osmoprotectants and antioxidants" is not a clear study. In materials and methods, authors have mentioned two levels of salinity (0 and 2%), but in all figures and tables, I cannot see anything related to normal and saline conditions. Add the data on rice under control conditions in comparison with saline conditions. However, there are some comments which can improve this article.

Response: Thanks for the comments. There have been two experiments in this study. To improve the clarity, we revised the section in the Materials and methods as "In this study two experiments were conducted. In Experiment I, there were two treatments: a control (CK) and a 0.2% salt stress treatment. Prior to transplanting, sea salt (Blue Starfish Salt Product Co., Ltd., Zhejiang, China) was added to the pots. For the CK and salt stress treatments (0.2%), 0 g and 26 g of salt were applied, respectively. In experiment II, for the salt stress treatment (0.2%), four concentrations of exogenous GABA (0 μM, 50 μM, 100 μM, and 150 μM) were sprayed to assess its effects on plant growth. All the spray solutions contained Tween-20 of a final concentration of 0.02% (v/v) as a surfactant. GABA was applied to both sides of the leaves during the pre-tillering and jointing stages manually. Spraying was done every two days for a total of seven days at 18:00. For the salt treatment, a soil salinity meter (TR-6D, Shunkeda, Beijing, China) was used to monitor and maintain stable soil salinity during the plant growth period (Table 1)." (Line 123-Line 133). That is why there were no data was presented related to the comparison between normal and saline conditions, except for Figure 1. 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, You have made significant changes now this can be accepted in this form.