Study on the Mechanism of Exogenous 5-Aminolevulinic Acid (ALA) in Regulating the Photosynthetic Efficiency of Pear Leaves

Round 1

Reviewer 1 Report

This study demonstrated the upregulation of key genes related to ALA biosynthesis and chlorophyll synthesis in pear leaves. Investigation of the effect of ALA on photosynthetic gas exchange parameters and gene expression provides valuable insights into enhancing plant photosynthetic efficiency. The experimental methods employed, such as GUS staining and promoter sequence cloning, are crucial for elucidating the mechanisms underlying ALA regulation in pear leaves. The language used is scientific and technical and is appropriate for the target audience. However, some complex terms or abbreviations can be explained or defined by readers who may not be familiar with them. Please check for typographical errors and inconsistencies in terminology throughout the manuscript. The following are some comments that need to be considered to improve the manuscript.

 Abstract:

In lines 15-18, consider breaking the sentence into two for easier comprehension: "ALA promoted the expression of genes encoding PSII reaction center proteins, such as core protein D1, inner light-harvesting pigment proteins CP43 and CP47, and cytochrome b559. This led to increased PSII reaction center activity."

Lines 18-20, simplify the sentence: "ALA alleviated donor side oxygen-evolving complex inhibition and reduced the closure rate on the receptor side, allowing for increased photochemical electron transfer, reduced heat dissipation, and improved photosynthetic performance indices PIabs and PI total."

Lines 20-21, consider rephrasing the conclusion: "The findings of this study contribute to a better understanding of ALA's role in promoting plant photosynthetic efficiency, providing valuable insights for further research and potential applications in pear production."

Introduction: The introduction part provides a good background on the importance of pear cultivation in China and highlights the relationship between photosynthesis and fruit quality. Here are some suggestions to improve the clarity and flow of the introduction: By addressing some suggestions, the introduction will be more concise and clear, providing a strong foundation for the rest of your manuscript.

Lines 27-29, consider revising the sentence for better readability: "Pears are the second largest deciduous fruit tree in China after apples, with a total cultivated area of 921.61×1000 hm² in 2021, resulting in a total output of 19.88 million tons."

Lines 31-32, simplify the sentence: "Although the pear cultivation area has slightly decreased due to national policy adjustments, the total output continues to grow as a result of increased unit yield."

Lines 34-35, rephrase the sentence for clarity: "Improper management practices, such as the overuse of inorganic fertilizers to compensate for insufficient organic fertilizer, can lead to fruit quality issues."

Lines 37-38, correct the typo: "H₂O₂" should be "H₂O". Additionally, consider breaking the sentence into two for easier comprehension: "Photosynthesis is a process of matter and energy conversion in plants. It involves the photolysis of H₂O to release oxygen, the conversion of solar energy into electrochemical energy, and the transformation of atmospheric CO₂ into carbohydrates to store energy in organic compounds."

Lines 57-58, break the sentence into two for better readability: "ALA application in pear production has been reported to have various effects, such as flower thinning, improved fruit quality, and enhanced anthocyanin accumulation."

Lines 62-63, consider revising the sentence: "Despite the potential benefits, the mechanisms behind ALA's effects on pear biological processes are not yet fully understood."

Lines 91-92, consider revising the sentence for better flow: "Our team recently reported that ALA promotes peach leaf photosynthesis and photosynthate transport from source to sink, contributing to high-quality fruit production."

Lines 97-98, simplify the sentence: "Spraying ALA solution at the late blooming stage can thin flowers, reducing the need for costly fruit thinning."

Lines 103-105, consider breaking the sentence into two for better readability: "To further investigate ALA's effects on photosynthetic capacity in pear trees, we conducted a study involving ALA solution application at specific growth stages, including root irrigation before flowering and flower spraying at the end of the flowering stage."

Lines 107-108, clarify the sentence: "We analyzed leaf gas exchange, chlorophyll fast fluorescence, and the expression of genes related to chlorophyll synthesis and photosynthetic electron transport carriers."

Other comments:

Rubisco: Abbreviations are usually defined at the first use in the as well as in the main text. please check whether 'Rubisco' should be defined before? Please check throughout the MS. Similarly other abbreviations throughout the manuscript.

Materials and Methods: The Materials and Methods section provides a detailed description of the experimental setup and the procedures followed during the study. Information regarding the experimental location, plant materials, treatments, and methods used is comprehensive and clearly outlined. The Materials and Methods section is comprehensive and provides a detailed overview of the experimental procedures. Clarifying specific details, providing references for formulas, and including additional information on controls and data analysis would further enhance the quality and reproducibility of this study.

1. It is important to ensure that all specific details related to the experiment, such as the concentration of solutions used, timing of treatments, and equipment specifications, are accurately reported by other researchers to allow for reproducibility.

2. The determination of pigment content in leaves, including the formulas used for calculating chlorophyll a, chlorophyll b, and carotenoid content, is well documented. However, it would be beneficial to provide references to the formulas cited in the text.

3. Methods for determining endogenous ALA content, ALA biosynthesis, and metabolism in leaves have been clearly described. Including specific details, such as centrifugation speed, incubation conditions, and measurement techniques, enhanced the reproducibility and validity of the results.

4. The procedures for measuring the photosynthetic gas exchange parameters and chlorophyll fast fluorescence are well explained. It is essential to include information on the calibration procedures, controls, and data analysis methods to ensure the accuracy and reliability of the measurements.

5. RNA extraction and RT-qPCR analysis methods are detailed and follow standard protocols. It is important to provide information on the selection of reference genes, primer design validation (note that the primer sequence mentioned in the main manuscript should be added to the supplementary data), and statistical analysis methods used for gene expression quantification. What does mean of “gDNA” genomic DNA?

6. The construction of plant gene expression vectors and subsequent transformation steps has been well documented. It would be beneficial to include information on vector design rationale, transformation efficiency calculations, and quality control measures for vector construction. Please be careful with italic fonts and scientific nomenclature of species and genera; for example, Agrobacterium throughout the MS is the same it should be italicized to keep the scientific pattern.

Results:

1. To maintain the scientific vibration, 3. Results and analysis into “Results” word only.

2. These results provide a detailed overview of the effects of exogenous ALA treatment on photosynthetic gas exchange parameters and chlorophyll fluorescence characteristics in pear leaves. This information is well-structured and presented in a logical sequence. It would be beneficial to include a brief introductory paragraph at the beginning of the Results section to provide an overview of the key findings before delving into specific details.

3. Ensure that all figures and tables are properly referenced and discussed in the text to provide context and interpretation for readers.

4. please check carefully if figure legends are included and appear consecutively in the manuscript.

Discussion

Overall, this discussion provides a detailed analysis of the effects of exogenous ALA treatment on the photosynthetic efficiency of pear leaves. This discussion provides a clear and structured analysis of experimental results. The flow of information is logical and easy to follow, which enhances the text’s overall readability. The following are some comments from the discussion.

1. This discussion does a good job of interpreting the results obtained from the experiments. However, it would be helpful to provide more in-depth explanations of the mechanisms underlying the observed effects of ALA on various parameters related to photosynthesis. Any schematic/hypothetical pathway or mechanism addressed any unexpected or contradictory results observed during the study, providing possible explanations or hypotheses for further investigation.

2. Citation and Attribution: This discussion effectively integrates previous research findings to support the results of the current study. However, it would be beneficial to provide more direct citations for specific claims and findings, particularly when referring to previous studies.

3. It would be beneficial to discuss the implications of these findings in the broader context of plant physiology and potential applications in agriculture or horticulture.

4. Considering the promising results obtained in this study, it would be beneficial to suggest future research directions. Identifying specific unanswered questions or areas that require further exploration could guide future research in this field.

References: Please check references in the text and follow the journal format. I found some references cited in the introduction section with only et al., (without years quotation). Moreover, overall references are enough to support the results and discussion. 

Moderate editing of the English language is required. 

Author Response

Comments and Suggestions for Author 1

This study demonstrated the upregulation of key genes related to ALA biosynthesis and chlorophyll synthesis in pear leaves. Investigation of the effect of ALA on photosynthetic gas exchange parameters and gene expression provides valuable insights into enhancing plant photosynthetic efficiency. The experimental methods employed, such as GUS staining and promoter sequence cloning, are crucial for elucidating the mechanisms underlying ALA regulation in pear leaves. The language used is scientific and technical and is appropriate for the target audience. However, some complex terms or abbreviations can be explained or defined by readers who may not be familiar with them. Please check for typographical errors and inconsistencies in terminology throughout the manuscript. The following are some comments that need to be considered to improve the manuscript.

R: Thank you very much for your kind comments and suggestions. We have checked the manuscript carefully and revised the mistakes.

Abstract:

In lines 15-18, consider breaking the sentence into two for easier comprehension: "ALA promoted the expression of genes encoding PSII reaction center proteins, such as core protein D1, inner light-harvesting pigment proteins CP43 and CP47, and cytochrome b559. This led to increased PSII reaction center activity."

R: Thank you. We have accepted your suggestion.

Lines 18-20, simplify the sentence: "ALA alleviated donor side oxygen-evolving complex inhibition and reduced the closure rate on the receptor side, allowing for increased photochemical electron transfer, reduced heat dissipation, and improved photosynthetic performance indices PIabs and PI total."

R: Thank you. We have accepted your suggestion.

Lines 20-21, consider rephrasing the conclusion: "The findings of this study contribute to a better understanding of ALA's role in promoting plant photosynthetic efficiency, providing valuable insights for further research and potential applications in pear production."

R: Thank you. We have accepted your suggestion.

Introduction: The introduction part provides a good background on the importance of pear cultivation in China and highlights the relationship between photosynthesis and fruit quality. Here are some suggestions to improve the clarity and flow of the introduction: By addressing some suggestions, the introduction will be more concise and clear, providing a strong foundation for the rest of your manuscript.

R: Thank you for your kind suggestion.

Lines 27-29, consider revising the sentence for better readability: "Pears are the second largest deciduous fruit tree in China after apples, with a total cultivated area of 921.61×1000 hm² in 2021, resulting in a total output of 19.88 million tons."

R: Thank you. We have accepted your suggestion.

Lines 31-32, simplify the sentence: "Although the pear cultivation area has slightly decreased due to national policy adjustments, the total output continues to grow as a result of increased unit yield."

R: Thank you. We have accepted your suggestion.

Lines 34-35, rephrase the sentence for clarity: "Improper management practices, such as the overuse of inorganic fertilizers to compensate for insufficient organic fertilizer, can lead to fruit quality issues."

R: Thank you. We have accepted your suggestion.

Lines 37-38, correct the typo: "H₂O₂" should be "H₂O". Additionally, consider breaking the sentence into two for easier comprehension: "Photosynthesis is a process of matter and energy conversion in plants. It involves the photolysis of H₂O to release oxygen, the conversion of solar energy into electrochemical energy, and the transformation of atmospheric CO₂ into carbohydrates to store energy in organic compounds."

R: Thank you. We have modified "H₂O₂" to "H₂O”. Also, we have accepted your other suggestion.

Lines 57-58, break the sentence into two for better readability: "ALA application in pear production has been reported to have various effects, such as flower thinning, improved fruit quality, and enhanced anthocyanin accumulation."

R: Thank you. We have accepted your suggestion.

Lines 62-63, consider revising the sentence: "Despite the potential benefits, the mechanisms behind ALA's effects on pear biological processes are not yet fully understood." 

R: Thank you. We have accepted your suggestion.

Lines 91-92, consider revising the sentence for better flow: "Our team recently reported that ALA promotes peach leaf photosynthesis and photosynthate transport from source to sink, contributing to high-quality fruit production."

R: Thank you. We have generally accepted your suggestion.

Lines 97-98, simplify the sentence: "Spraying ALA solution at the late blooming stage can thin flowers, reducing the need for costly fruit thinning."

R: Thank you. We have accepted your suggestion.

Lines 103-105, consider breaking the sentence into two for better readability: "To further investigate ALA's effects on photosynthetic capacity in pear trees, we conducted a study involving ALA solution application at specific growth stages, including root irrigation before flowering and flower spraying at the end of the flowering stage."

R: Thank you. We have accepted your suggestion.

Lines 107-108, clarify the sentence: "We analyzed leaf gas exchange, chlorophyll fast fluorescence, and the expression of genes related to chlorophyll synthesis and photosynthetic electron transport carriers."

R: Thank you. We have accepted your suggestion.

Other comments:

Rubisco: Abbreviations are usually defined at the first use in the as well as in the main text. please check whether 'Rubisco' should be defined before? Please check throughout the MS. Similarly other abbreviations throughout the manuscript.

R: Thank you very much. We've added definitions for words like PSII, PSI, PP2A, and SnRK2.6 by providing full definitions when the acronym first appeared and checking the full text.

Materials and Methods: The Materials and Methods section provides a detailed description of the experimental setup and the procedures followed during the study. Information regarding the experimental location, plant materials, treatments, and methods used is comprehensive and clearly outlined. The Materials and Methods section is comprehensive and provides a detailed overview of the experimental procedures. Clarifying specific details, providing references for formulas, and including additional information on controls and data analysis would further enhance the quality and reproducibility of this study.

R: Thank you. We have checked it carefully.

1. It is important to ensure that all specific details related to the experiment, such as the concentration of solutions used, timing of treatments, and equipment specifications, are accurately reported by other researchers to allow for reproducibility.

R: Thank you. We have checked it carefully.

2. The determination of pigment content in leaves, including the formulas used for calculating chlorophyll a, chlorophyll b, and carotenoid content, is well documented. However, it would be beneficial to provide references to the formulas cited in the text.

R: Thank you. We have checked it carefully. These is a reference in the text.

Wang, X.K. Experimental Principles and Techniques of Plant Physiology and Biochemistry. Beijing: Higher Education Press 2006, 130-134.

3. Methods for determining endogenous ALA content, ALA biosynthesis, and metabolism in leaves have been clearly described. Including specific details, such as centrifugation speed, incubation conditions, and measurement techniques, enhanced the reproducibility and validity of the results.

R: Thank you for your kind comment.

4. The procedures for measuring the photosynthetic gas exchange parameters and chlorophyll fast fluorescence are well explained. It is essential to include information on the calibration procedures, controls, and data analysis methods to ensure the accuracy and reliability of the measurements.

R: Thank you for your kind comment.

5. RNA extraction and RT-qPCR analysis methods are detailed and follow standard protocols. It is important to provide information on the selection of reference genes, primer design validation (note that the primer sequence mentioned in the main manuscript should be added to the supplementary data), and statistical analysis methods used for gene expression quantification. What does mean of “gDNA” genomic DNA?

R: Thank you for your kind comment. “gDNA” has been modified to “genomic DNA”.

6. The construction of plant gene expression vectors and subsequent transformation steps has been well documented. It would be beneficial to include information on vector design rationale, transformation efficiency calculations, and quality control measures for vector construction.Please be careful with italic fonts and scientific nomenclature of species and genera; for example, Agrobacterium throughout the MS is the same it should be italicized to keep the scientific pattern.

R: Thank you. We have checked it carefully. You suggested that we’d better include information on vector design rationale, transformation efficiency calculations, and quality control measures for vector construction. However, we performed the experiments according to instruction of the kits, and have not systematically calculated the specific efficiency. Therefore, we cannot add more.

Results:

1. To maintain the scientific vibration, 3. Results and analysis into “Results” word only.

R: Thank you for your kind comment. “Results and analysis” have been modified to “Results”.

2. These results provide a detailed overview of the effects of exogenous ALA treatment on photosynthetic gas exchange parameters and chlorophyll fluorescence characteristics in pear leaves. This information is well-structured and presented in a logical sequence. It would be beneficial to include a brief introductory paragraph at the beginning of the Results section to provide an overview of the key findings before delving into specific details.

R: Thank you for your kind comment. You suggested to add a brief introductory paragraph at the beginning of the Results section to provide an overview of the key findings before delving into specific details. Mr. Wang QX had written a paragraph. However, I feel it strange and delete it. In most situations, I’d like to exhibit our results gradually. I think it better for readers to understand.

3. Ensure that all figures and tables are properly referenced and discussed in the text to provide context and interpretation for readers.

R: Thank you very much. We have checked it carefully.

4. please check carefully if figure legends are included and appear consecutively in the manuscript.

R: Thank you very much. We have checked it carefully.

Discussion

Overall, this discussion provides a detailed analysis of the effects of exogenous ALA treatment on the photosynthetic efficiency of pear leaves. This discussion provides a clear and structured analysis of experimental results. The flow of information is logical and easy to follow, which enhances the text’s overall readability. The following are some comments from the discussion.

R: Thank you for your kind comment.

1. This discussion does a good job of interpreting the results obtained from the experiments. However, it would be helpful to provide more in-depth explanations of the mechanisms underlying the observed effects of ALA on various parameters related to photosynthesis. Any schematic/hypothetical pathway or mechanism addressed any unexpected or contradictory results observed during the study, providing possible explanations or hypotheses for further investigation.

R: Thank you for your kind comment. However, just as you know, photosynthesis is a complex biochemical process, which has not been completely known. Similarly, ALA has various functions in plant production, most of the regulatory mechanisms is also not clear, which has been discussed in the previous review (Wang et al., 2023, Forests, 14, 1857. https://doi.org/10.3390/f14091857). We're working in this direction. It can be believed not so far that we will understand the mechanism of ALA regulating photosynthetic capacity in plants much more than it does today.

2. Citation and Attribution: This discussion effectively integrates previous research findings to support the results of the current study. However, it would be beneficial to provide more direct citations for specific claims and findings, particularly when referring to previous studies.

R: Thank you very much. We tried to improve our manuscript according to your suggestions.

3. It would be beneficial to discuss the implications of these findings in the broader context of plant physiology and potential applications in agriculture or horticulture.

R: Thank you very much. We tried to improve our manuscript according to your suggestions.

4. Considering the promising results obtained in this study, it would be beneficial to suggest future research directions. Identifying specific unanswered questions or areas that require further exploration could guide future research in this field.

R: Thank you very much. We tried to improve our manuscript according to your suggestions.

References: Please check references in the text and follow the journal format. I found some references cited in the introduction section with only et al., (without years quotation). Moreover, overall references are enough to support the results and discussion.

R: Thank you for your kind reminds. We have added years in the text.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors investigated the effect of exogenous 5-aminolevulinic acid (ALA) on photosynthetic pigments, photosynthetic gas exchange parameters, chlorophyll fast fluorescence, and the expression of related genes. Additionally, the authors extracted a few important promoters related to photosynthesis and cloned them with the GUS gene using a plant transformation vector. Subsequently, they studied their activity in agro-infiltrated tobacco leaves through GUS staining and quantified the expression of the GUS gene.

Comments:

1. The manuscript is well-written and easy to read, with supportive images. However, the main issue with the abstract is clarity regarding the experiments conducted in pear plants versus tobacco leaves using Agro-infiltration/injection. In the abstract, the phrase “transformation into chlorophylls” should be revised to “enhancement of chlorophyll content”. Additionally, it is unclear how the authors claim that ALA activated the promoter activity of PypHEMA and PypCHLH genes (necessary genes to initiate Chl biosynthesis) associated directly with promoting the synthesis of endogenous ALA (Line no 15). Furthermore, the authors should clarify that promoters were isolated and cloned with GUS genes and their activity was tested in agroinfiltrated tobacco leaves to improve clarity. In its current form, the abstract does not clearly distinguish between experiments conducted in pear and those conducted in tobacco, necessitating careful rewriting.

2. Table 1 does not mention the source from where the primers are designed, rendering it less useful for readers or researchers who wish to follow the research. I suggest providing Genebank numbers from where the primers are designed. Authors can list the Genebank numbers, followed by the primers (both forward and reverse in the same column, labelled as Fw: and Rv: respectively), and then the amplicon size. Additionally, indicate whether the primers are for gene or promoter sequences. Please provide dissociation or melting curves for each primer as a supplementary file. Dissociation curves are crucial for quality assessment and would not only demonstrate data quality but also assist researchers in selecting primers from your study.

3. Provide the full form of ALA in the title and provide the full name when the abbreviation is used for the first time (Line no 7).

4. Provide details for the ALA solution, specifying what "5L containing 10MG/L ALA" means.

5.    Edit the footnote of Table 2 to state "15 biological replicates".

Author Response

Comments and Suggestions for Author 2

The authors investigated the effect of exogenous 5-aminolevulinic acid (ALA) on photosynthetic pigments, photosynthetic gas exchange parameters, chlorophyll fast fluorescence, and the expression of related genes. Additionally, the authors extracted a few important promoters related to photosynthesis and cloned them with the GUS gene using a plant transformation vector. Subsequently, they studied their activity in agro-infiltrated tobacco leaves through GUS staining and quantified the expression of the GUS gene.

R: Thank you for your kind comment.

Comments:

1. The manuscript is well-written and easy to read, with supportive images. However, the main issue with the abstract is clarity regarding the experiments conducted in pear plants versus tobacco leaves using Agro-infiltration/injection. In the abstract, the phrase “transformation into chlorophylls” should be revised to “enhancement of chlorophyll content”. Additionally, it is unclear how the authors claim that ALA activated the promoter activity ofPypHEMA and PypCHLH genes (necessary genes to initiate Chl biosynthesis) associated directly with promoting the synthesis of endogenous ALA (Line no 15). Furthermore, the authors should clarify that promoters were isolated and cloned with GUS genes and their activity was tested in agroinfiltrated tobacco leaves to improve clarity. In its current form, the abstract does not clearly distinguish between experiments conducted in pear and those conducted in tobacco, necessitating careful rewriting.

R: Thank you for your kind suggestion. We have modified the abstract.

2. Table 1 does not mention the source from where the primers are designed, rendering it less useful for readers or researchers who wish to follow the research. I suggest providing Genebank numbers from where the primers are designed. Authors can list the Genebank numbers, followed by the primers (both forward and reverse in the same column, labelled as Fw: and Rv: respectively), and then the amplicon size. Additionally, indicate whether the primers are for gene or promoter sequences. Please provide dissociation or melting curves for each primer as a supplementary file. Dissociation curves are crucial for quality assessment and would not only demonstrate data quality but also assist researchers in selecting primers from your study.

R: Thank you for your kind suggestion. We have modified Table 1. And in the text, we added gene or promoter sequences from NCBI. However, the melting curves are generally not presented in manuscript, which is present in a supplementary table here.

3. Provide the full form of ALA in the title and provide the full name when the abbreviation is used for the first time (Line no 7).

R: Thank you. We have accepted your suggestion.

4. Provide details for the ALA solution, specifying what "5L containing 10MG/L ALA" means.

R: Thank you. We have accepted your suggestion.

5. Edit the footnote of Table 2 to state "15 biological replicates".

R: Thank you. We have accepted your suggestion.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript "Study on the mechanism of exogenous ALA in regulating the photosynthetic efficiency of pear leaves" by Xinqing Wang, Jianting Zhang, and Liangju Wang represents a fundamental study of the effects of aminolevulinic acid on the photosynthetic apparatus of peach plants.

An integrated approach and, accordingly, different research methods were used to solve the problems posed in the study.

The design of the experiments, the reliability of the data and their presentation raise no questions.

The only thing that I would like to further clarify is the analysis of the primary causes of the observed effects: the action of ALA as a regulator of the expression of chlorophyll synthesis genes or the metabolic effects of ALA.

Author Response

Comments and Suggestions for Author 3

The manuscript "Study on the mechanism of exogenous ALA in regulating the photosynthetic efficiency of pear leaves" by Xinqing Wang, Jianting Zhang, and Liangju Wang represents a fundamental study of the effects of aminolevulinic acid on the photosynthetic apparatus of peach plants.

An integrated approach and, accordingly, different research methods were used to solve the problems posed in the study.

The design of the experiments, the reliability of the data and their presentation raise no questions.

The only thing that I would like to further clarify is the analysis of the primary causes of the observed effects: the action of ALA as a regulator of the expression of chlorophyll synthesis genes or the metabolic effects of ALA.

R: Thank you for reading and pointing this out. I explain this question as follows. As a precursor of chlorophyll synthesis, ALA can widely and effectively promote chlorophyll biosynthesis and photosynthetic electron transport under both non-stress and stress conditions, and ALA can participate in the regulation of chlorophyll synthesis gene expression. At the same time, ALA has many metabolites in organisms. From the present study, exogenous ALA can effectively promote endogenous ALA synthesis and its metabolite production. We cannot exclude the possibility that ALA metabolites are also involved in the regulation of chlorophyll synthesis gene expression. In conclusion, ALA can regulate chlorophyll biosynthesis, whether it is its own function or the involvement of its metabolites, the present study cannot give a definite conclusion, and the issue still needs further research.

Author Response File: Author Response.pdf