Microbial–Organic Inputs with Glycine Supplementation Enhance Growth and Heat Stress Tolerance in Lettuce

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

General comments

I have read the manuscript “Microbial Consortium and Organic Amendments Enhance Growth and High-Temperature Stress Tolerance in Lettuce”. Authors investigate the lettuce growth attributes, photosynthetic parameters, phenolic and flavonoids, antioxidant activities adaptability under high temperature stress. This research is doing well and this research is obvious application potential to address the problems of those farmers who are suffering from the low growth and yield due to high temperature stress. In this sense the manuscript is very valuable. However, I found some lacking information especially in the introduction and discussion section. I found the lack of potential and the latest reference to support their findings. I request to author for Major revision of this manuscript.

1. need to explain study site in the abstract
2. add future perspectives in the abstract
3. L39 compounded is not suitable word, change
4. Need to further explain the molecular mechanism of upregulation of heat shock proteins (HSPs) and transcription factors (TFs) that modulate stress-responsive gene expression under heat stress
5. Overall introduction is well written, but the argument needs to link with heat stress with proper citations
6. Need to explain the Biostimulants and PGPR potential mechanisms in improving nitrogen assimilation, maintaining ionic balance, and boosting antioxidant defenses under drought stress.
7. Improve research gaps, and research objectives
8. L138-140 Need to address soil physicochemical properties which was used to fill-up pots
9. The captions of the figures need to be revised and explain treatments and abbrevations.
10. The figures lack P value, revise and address P value in each fig
11. Lacks potential relationships among observed indices, I suggest you to draw, PCA and Pearson Correlation Analysis
12. Need to improve discussion by linking mechanisms to heat stress mitigation by obtained results
13. Make a clear comparison of your obtained results with clear justification regarding 30- and 60-days stress conditions
14. add study limitation, practical implications and future perspectives
15. Revise this statement “Overall, this work contributes to the growing body of evidence supporting microbiome-based bioformulations as a cornerstone of next-generation sustainable agriculture”

 

 

Comments on the Quality of English Language

English language is good, but futher sentense structure can be improved for more clearity

Author Response

Comments 1: need to explain study site in the abstract
Response 1: We have added a brief description of the study site location in the abstract to provide better context for the experimental setup. [Lines 16-18]

Comments 2: add future perspectives in the abstract
Response 2: We have added a sentence at the end of the abstract that highlights the potential future applications and research directions stemming from our findings. [Lines 28-30]

Comments 3: L39 compounded is not suitable word, change
Response 3: We have replaced the word "compounded" with “exacerbated” to improve clarity and precision. [Line 39]

Comments 4: Need to further explain the molecular mechanism of upregulation of heat shock proteins (HSPs) and transcription factors (TFs) that modulate stress-responsive gene expression under heat stress
Response 4: We have elaborated on the molecular mechanisms by which HSPs and TFs modulate plant stress responses under heat stress, supported by relevant citations. [Lines 55-62]

Comments 5: Overall introduction is well written, but the argument needs to link with heat stress with proper citations
Response 5: We have strengthened the connection between biostimulant/PGPR application and heat stress mitigation by incorporating relevant references and expanding the argument accordingly. [Lines 65-79 and Lines 80-89]

Comments 6: Need to explain the Biostimulants and PGPR potential mechanisms in improving nitrogen assimilation, maintaining ionic balance, and boosting antioxidant defenses under drought stress.
Response 6: We have added the mechanisms through which biostimulants and PGPR improve nitrogen assimilation, ionic homeostasis, and antioxidant defenses under abiotic stress. [Lines 80-89]

Comments 7: Improve research gaps, and research objectives
Response 7: We have revised the final paragraph of the introduction to clearly articulate the research gaps and explicitly state the research objectives of the study. [Lines 92-119]

Comments 8: L138-140 Need to address soil physicochemical properties which was used to fill-up pots
Response 8: We have now included information on the physicochemical properties of the soil used in the pots to ensure reproducibility and provide context for the experimental conditions. [Lines 149-154]

Comments 9: The captions of the figures need to be revised and explain treatments and abbrevations.
Response 9: All figure captions have been revised to clearly describe the experimental treatments and define all abbreviations to enhance reader comprehension.

Comments 10: The figures lack P value, revise and address P value in each fig
Response 10: We have revised all figures to include statistical annotations (e.g., asterisks for significance levels) and provided exact P values from Kruskal-Wallis.

Comments 11: Lacks potential relationships among observed indices, I suggest you to draw, PCA and Pearson Correlation Analysis
Response 11: To uncover potential relationships among physiological and biochemical traits, we have performed Principal Component Analysis (PCA) and Pearson correlation analysis. [Lines 255-259, 400-442, and 463-478]

Comments 12: Need to improve discussion by linking mechanisms to heat stress mitigation by obtained results
Response 12: In the revised discussion section, we have explicitly linked the observed physiological and biochemical responses to heat stress mitigation. We highlighted how PYS-based treatments enhance antioxidant enzyme activities (e.g., APX, CAT, POD), reduce lipid peroxidation, and increase the accumulation of osmoprotectants like glycine betaine—all of which contribute to improved redox homeostasis and cellular stability under heat stress. [Lines 592-606]

Comments 13: Make a clear comparison of your obtained results with clear justification regarding 30- and 60-days stress conditions
Response 13: We have provided a clearer comparison between the 30-day and 60-day time points. Specifically, we describe how early responses, such as increased lipid peroxidation and antioxidant activation at 30 days, likely reflect an initial ROS burst and stress perception, while the reduction in oxidative damage and increased secondary metabolite accumulation at 60 days indicate successful stress adaptation and priming effects. [Lines 592-606]

Comments 14: add study limitation, practical implications and future perspectives
Response 14: We have added study limitations, practical applications in tropical agriculture, and directions for future research. [Lines 601-606 and 622-627]

Comments 15: Revise this statement “Overall, this work contributes to the growing body of evidence supporting microbiome-based bioformulations as a cornerstone of next-generation sustainable agriculture”
Response 15: We have rephrased the sentence. [Lines 622-627]

Reviewer 2 Report

Comments and Suggestions for Authors

Title:

Microbial Consortium and Organic Amendments Enhance Growth and High-Temperature Stress Tolerance in Lettuce

 

Recommendation:

Minor revision.

 

Comments:

This study investigates the use of microbial consortia combined with organic fertilizers, growth regulators, and bio-chemical supplements to enhance lettuce growth, stress tolerance, and quality under high-temperature conditions. The study evaluates how these natural treatments can improve plant biomass, photosynthetic pigments, antioxidant defenses, and osmoprotectant accumulation, ultimately aiming to reduce reliance on chemical fertilizers and promote sustainable agriculture practices. The subject is relevant and consistent with the aims and scopes of the journal. Some comments and suggestions are offered below with the intent to assist the author in improving the manuscript.

 

1. There is a formatting error in the section numbering. Specifically, the manuscript contains two sections labeled "2.2" and two labeled "2.4.6". Therefore, the entire subsection numbering in Section 2 needs to be carefully reviewed and corrected.
2. This study reports some very interesting findings. However, it would be valuable to know whether the authors have considered or discussed the following question: What are the underlying molecular mechanisms by which the PYS microbial consortium enhances antioxidant defense and osmoprotectant synthesis in lettuce?
3. In this study, the authors noted that Vitamin C levels were lower in PYS-treated plants and proposed that a metabolic trade-off might favor phenylpropanoid pathway activation. In light of this, I offer two suggestions for the authors to consider during revision: (1) Consider citing supporting literature or offering hypotheses, such as competition for metabolic precursors (e.g., glucose or ascorbate-related intermediates). (2) Briefly explain the potential trade-off between ascorbate and phenolic biosynthesis to strengthen the rationale for this observation.
4. In this study, the authors mentioned that the PYS+OF+Gly treatment showed increased lipid peroxidation at 60 days, yet total biomass was still significantly higher. This apparent contradiction, enhanced physiological stress without a corresponding reduction in growth, raises some important considerations. I suggest the following two points for possible revision: (1) Discuss the possibility that glycine may play a dual role, either promoting growth or exacerbating oxidative stress depending on the developmental stage. (2) Consider whether environmental factors (e.g., temperature fluctuations or pot-based growth conditions) may have influenced certain physiological responses, to avoid over-attributing effects solely to the treatments.

Author Response

Comments 1: There is a formatting error in the section numbering. Specifically, the manuscript contains two sections labeled "2.2" and two labeled "2.4.6". Therefore, the entire subsection numbering in Section 2 needs to be carefully reviewed and corrected.
Response 1: We have thoroughly reviewed and corrected the section and subsection numbering in Section 2 to ensure consistency and avoid repetition. The corrected numbering has been implemented throughout the manuscript.

Comments 2: This study reports some very interesting findings. However, it would be valuable to know whether the authors have considered or discussed the following question: What are the underlying molecular mechanisms by which the PYS microbial consortium enhances antioxidant defense and osmoprotectant synthesis in lettuce?
Response 2: While our study focused primarily on physiological and biochemical responses, we have incorporated a paragraph addressing potential molecular mechanisms. We now propose that components of the PYS consortium—such as Methylobacterium, Sphingomonas, and Acinetobacter—may produce IAA, VOCs, and exopolysaccharides that activate MAPK signaling and transcription factors. These regulators are known to control the expression of genes involved in ROS detoxification. This mechanistic hypothesis is grounded in existing literature and provides a foundation for future molecular studies. [Lines 579-591]

Comments 3: In this study, the authors noted that Vitamin C levels were lower in PYS-treated plants and proposed that a metabolic trade-off might favor phenylpropanoid pathway activation. In light of this, I offer two suggestions for the authors to consider during revision: (1) Consider citing supporting literature or offering hypotheses, such as competition for metabolic precursors (e.g., glucose or ascorbate-related intermediates). (2) Briefly explain the potential trade-off between ascorbate and phenolic biosynthesis to strengthen the rationale for this observation.
Response 3: We have added a mechanistic explanation of the metabolic trade-off between the ascorbate and phenylpropanoid pathways, supported by literature on carbon partitioning and shared precursor competition. [Lines 520-533]

Comments 4: In this study, the authors mentioned that the PYS+OF+Gly treatment showed increased lipid peroxidation at 60 days, yet total biomass was still significantly higher. This apparent contradiction, enhanced physiological stress without a corresponding reduction in growth, raises some important considerations. I suggest the following two points for possible revision: (1) Discuss the possibility that glycine may play a dual role, either promoting growth or exacerbating oxidative stress depending on the developmental stage. (2) Consider whether environmental factors (e.g., temperature fluctuations or pot-based growth conditions) may have influenced certain physiological responses, to avoid over-attributing effects solely to the treatments.
Response 4: We have added a discussion of glycine’s dual role in stress physiology and highlighted the influence of environmental factors such as temperature fluctuations and pot-based conditions as potential contributors to physiological variation. [Lines 538-548]

Reviewer 3 Report

Comments and Suggestions for Authors

The research presented by the authors is interesting. Performing such a large number of determinations is a huge amount of work. The manuscript was written in a logical and orderly manner, but it has several weaknesses.

1. The methodology lacks information on where the fertilizers used were purchased and more detailed information on what kind of fertilizers were used.
2. Can the authors explain why there are such large standard deviations in the results for many of the determinations performed? For example, for the PYS+OF+Gly sample in the lipid peroxidation assay, the deviation is practically 100%. Doesn't such a large scatter of results make them unreliable?

Comments for author File: Comments.pdf

Author Response

Comments 1: The methodology lacks information on where the fertilizers used were purchased and more detailed information on what kind of fertilizers were used.
Response 1: We have revised the Materials and Methods section to include the sources and specific details of all fertilizers used in the study. [Lines 155-159]

Comments 2: Can the authors explain why there are such large standard deviations in the results for many of the determinations performed? For example, for the PYS+OF+Gly sample in the lipid peroxidation assay, the deviation is practically 100%. Doesn't such a large scatter of results make them unreliable?
Response 2: We appreciate the reviewer’s concern regarding the variability in some of our data, particularly for the PYS+OF+Gly treatment in the lipid peroxidation assay. The large standard deviations likely reflect high biological variability due to complex interactions between microbial metabolites, organic inputs, and plant responses under heat stress conditions. We have now acknowledged this limitation in the revised discussion and emphasized the importance of further controlled and field-based experiments to better understand such variability. Despite the high variation, the statistical analyses confirmed significant differences between treatments, and the trends were biologically consistent across replicates. [Lines 601-606]

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have incorporated my all suggestion. I am satisfied with all edits. I have no more suggestions. Manuscript can be accepted.

Author Response

Thank you very much for your constructive comments and suggestions to improve our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Accept in present form

Author Response

Thank you very much for your constructive comments and suggestions to improve our manuscript.