Investigating the Variation between Lignin Content and the Fracture Characteristics in Capsicum annuum Mutant Stems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors propose a manuscript entitled “Investigating the correlation between lignin content and the fracture characteristics in Capsicum annuum mutant stems”. This study investigated the biomechanical properties and lignin content of four Capsicum annuum lines, and analysized gene expression patterns of five key lignin biosynthetic pathway genes (PAL, C4H, 4CL, CCoAOMT, CAD) in these lines. The results are helpful to understanding the correlation between lignin content and stem rigidity in Capsicum annuum, and may be of reference value in pepper breeding.

However, there are many facets in the manuscript that need improvement, especially in the section Materials and Methods

1. In the Materials and Methods, it is necessary to indicate the growth period of the examined materials, that is, how many days of cultivation of seedlings after seed germination. This is crucial for the subsequent explanation of lignin content and related gene expression levels and patterns.

2. In the analysis of gene expression patterns associated with lignin synthesis, it is best to include the expression situation of five genes in the control material.

3. Please briefly describe the PCR protocol in L155-165.

4. The expression of lignin content in Line 130-131 is difficult to understand and needs to be rewritten.

5. Abbreviations in the Figures and Tables need to be indicated in the captions, such as G × Sr (Table 3).

6. Continued after Figure 3, it is suggested to analyze the correlation between lignin content and breaking force within same a seedling. This can help understand the distribution characteristics of lignin in various component in seedlings and evaluate their breeding value.

7. The format of the references does not meet the criterion of the journal.

Author Response

Dear Reviewer,

I sincerely appreciate the time and effort you have taken to thoroughly review our submitted article. Your thoughtful feedback and valuable suggestions are greatly appreciated, and we are thankful for your insightful remarks.

1. In the Materials and Methods, it is necessary to indicate the growth period of the examined materials, that is, how many days of cultivation of seedlings after seed germination. This is crucial for the subsequent explanation of lignin content and related gene expression levels and patterns.

As you suggested we added the exact time period from germination until the experiments. Please see it in line 124-125. Since this is a very intense growth period, it makes sense to examine stem sections that are quite close to each other.

2. In the analysis of gene expression patterns associated with lignin synthesis, it is best to include the expression situation of five genes in the control material.

Thank you very much for the important observation. The reason why the control gene expression values are not provided in the gene expression diagram is that we examined relative expression here and we compared the mutants’ gene expression profile to the ’Garai fehér’.

Please briefly describe the PCR protocol in L155-165.

Following your suggestion we added more detailed information about the qPCR protocol, please see line 175-179.

3. The expression of lignin content in Line 130-131 is difficult to understand and needs to be rewritten.

As you suggested we rewrote the related part of the Materials and Methods, please see it in line 135-143

4. Abbreviations in the Figures and Tables need to be indicated in the captions, such as G × Sr (Table 3).

As suggested we added the meaning of these letters, please see in line 252.

5. Continued after Figure 3, it is suggested to analyze the correlation between lignin content and breaking force within same a seedling. This can help understand the distribution characteristics of lignin in various component in seedlings and evaluate their breeding value.

Thank you very much for the valuable suggestion. It is indeed possible to analyze the values in this way as well, but we used the method presented in the article because this way the detectable differences are more visible.

6. The format of the references does not meet the criterion of the journal.

As you asked, we corrected the form of the references, please see line 361-469.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Investigating the correlation between lignin content and the fracture characteristics in Capsicum annuum mutant stems” provides interesting information about the lignin content, as well as the bending strength of Capsicum annuum mutant stems and of a commercial variety.

However, there are important information missing:

How many stems of each kind were analysed?

Regarding the lignin content as determined by the acetyl bromide method, how many replicas of the same specimen were performed?

The absorptivity (e) is a very important parameter for the results of lignin content, please state it.

Results of the lignin content determined by this method should be analysed carefully, including a comparison of the three locations within the same stem.

The pith, wood, and bark are three distinct tissues that comprise the stem. The lignin content that is determined will be influenced by any variation in the percentage of each. Pith contains a negligible amount of lignin, but the results are influenced by protein. The wood's results are most precise when the material is extractive-free; otherwise, they are also influenced. Extractable and non-extractive material, as well as suberin, are counfounding effects in bark.

So the 2% difference between the top (10%) and bottom (8%) parts of Garai F could be explained solely by any observable differences in the proportion of the three tissues mentioned above within the stem. The same can be said about the differences between stems (mutantes and GaryF.).

I believe that the manuscript would be enhanced by a diverse approach, such as a “variation of lignin content and...”, rather than a "correlation between lignin content and the fracture characteristics."

This is also corroborated by the lack of correlation, as can be seen in Figure 3. Please note that for an 8% lignin content, the breaking force goes from almost zero to more than 30 N. If the approach is lignin vs breaking then it should not matter the stem part, shouldn’t it? Whatever the case, the coefficient of determination should always be presented!

Please change the lignin content form mg/g to %it will be clear that the number of decimal places used is too high for the precision of the methods!

Author Response

Dear Reviewer,

I sincerely appreciate the time and effort you have taken to thoroughly review our submitted article. Your thoughtful feedback and valuable suggestions are greatly appreciated, and we are thankful for your insightful remarks.

How many stems of each kind were analysed?

As you asked we added the detailed informations about the number of stems and the replicates, please see in line 137-138 and and 149-150.

Regarding the lignin content as determined by the acetyl bromide method, how many replicas of the same specimen were performed?

Following your suggestion we added the detailed informations about the number of stems and the replicates, please see in line 137-138.

The absorptivity (e) is a very important parameter for the results of lignin content, please state it.

We corrected this part by adding the missing information. To see the revised part please see line 139-142.

Results of the lignin content determined by this method should be analysed carefully, including a comparison of the three locations within the same stem.

The pith, wood, and bark are three distinct tissues that comprise the stem. The lignin content that is determined will be influenced by any variation in the percentage of each. Pith contains a negligible amount of lignin, but the results are influenced by protein. The wood's results are most precise when the material is extractive-free; otherwise, they are also influenced. Extractable and non-extractive material, as well as suberin, are counfounding effects in bark.

So the 2% difference between the top (10%) and bottom (8%) parts of Garai F could be explained solely by any observable differences in the proportion of the three tissues mentioned above within the stem. The same can be said about the differences between stems (mutantes and GaryF.).

Since the stem-breaking test treats the different tissues within the stem collectively, we also treat this chemical composition parameter as a whole in this analysis.

I believe that the manuscript would be enhanced by a diverse approach, such as a “variation of lignin content and...”, rather than a "correlation between lignin content and the fracture characteristics."

Thank you kindly for your suggestion, we made the correction in the title.

This is also corroborated by the lack of correlation, as can be seen in Figure 3. Please note that for an 8% lignin content, the breaking force goes from almost zero to more than 30 N. If the approach is lignin vs breaking then it should not matter the stem part, shouldn’t it? Whatever the case, the coefficient of determination should always be presented!

Thank you very much for the important observation. The reason it matters for the stem is that if we could focus this trait at the junction between the stem and the peduncle, it could be agronomically significant for harvesting. However, you are correct that if a brittle stem breaks at the base of the plant, it would be lethal for the plant, so this factor indeed becomes irrelevant.

Please change the lignin content form mg/g to %it will be clear that the number of decimal places used is too high for the precision of the methods!

As you suggested we changed the mg/g part to % in the whole manuscript. Please see the corrections in line 142, 189, 198, 202-220, 251.

Reviewer 3 Report

Comments and Suggestions for Authors

This study examines the relationship between lignin content and the mechanical properties, specifically the fracture characteristics, of mutant Capsicum annuum (pepper) stems. The researchers investigated three mutant lines: 'fragile-plant' (frx), 'tortuous internodi' (tti), and 'puffy-structured stem' (pfi), comparing them with a commercially established variety, 'Garai fehér'. Lignin content was measured using the Acetyl Bromide Method, while stem rigidity was assessed through three-point bending tests. Additionally, gene expression analysis was conducted to investigate the expression of key genes in the lignin biosynthetic pathway. The findings revealed significant differences in lignin content and breaking strength across the genotypes and stem regions. However, the correlation between lignin content and stem rigidity appears to be complex, with multiple factors influencing these traits.

The study employs a rigorous and comprehensive methodology, including the Acetyl Bromide Method for lignin quantification, three-point bending tests for mechanical property assessment, and qRT-PCR for gene expression analysis. This multi-faceted approach provides a thorough exploration of the factors influencing stem rigidity. The focus on mutant lines of Capsicum annuum and the investigation of their potential applicability in different cultivation methods is innovative. This research offers valuable insights into the relationship between lignin content and mechanical properties in horticultural plants, which could be of interest to breeding programmes.

The objectives of this study are clear, and the findings are of interest. While I do not have major criticisms, I have included some suggestions below:

The study aims to correlate lignin content with stem rigidity, but the results indicate that the relationship is not straightforward. The lack of a confirmed positive correlation between lignin content and mechanical properties in the mutant lines may somewhat weaken the overall conclusions. Providing additional evidence or further discussion could help clarify this point.

The gene expression analysis reveals the overexpression of certain lignin biosynthetic genes in the mutants. However, the anticipated correlation between gene expression and increased lignin content is not observed. This discrepancy suggests that the study may have overlooked other regulatory factors or mechanisms influencing lignin deposition.

While the study offers valuable insights into the mechanistic aspects of lignin content and stem rigidity, the discussion on the practical implications for breeding and cultivation is somewhat limited. Expanding on how these findings could be applied in agricultural practices would enhance the study's relevance to the field.

Comments on the Quality of English Language

Regarding the English writing, the manuscript is well-organised, with a clear structure that logically presents the research question, methods, results, and discussion. Each section transitions smoothly into the next, making the study easy to follow. However, some sentences are overly complex and could benefit from simplification. For example, long sentences with multiple clauses can be challenging to read and may obscure the main point. Simplifying these sentences would improve readability.

 

Line 18-20: Change "This study investigates the biomechanical properties and lignin content of various Capsicum annuum mutant lines, namely ‘fragile-plant’ (frx), ‘tortuous internodi’ (tti) ‘puffy-structured stem’ (pfi), and, compared to a commercially established variety, 'Garai fehér'." to "This study investigates the biomechanical properties and lignin content of various Capsicum annuum mutant lines—'fragile-plant' (frx), 'tortuous internodi' (tti), and 'puffy-structured stem' (pfi)—in comparison to a commercially established variety, 'Garai fehér'."

Line 37-38: Change "Horticulture, alongside crop production and livestock farming, represents one of the most crucial sectors within agriculture." to "Horticulture, alongside crop and livestock production, is one of the most crucial sectors in agriculture."

Line 70-71: Change "Lignin deficiency can lead to abnormal stem development, highlighting the complex interplay between genetics and biochemical processes in shaping plant architecture." to "Lignin deficiency can cause abnormal stem development, underscoring the complex interplay between genetics and biochemical processes in shaping plant architecture."

Line 205-206: Change "The correlation between lignin content and breaking strength in aspect of the various genotypes is illustrated in the following figure using a linear regression." to "The correlation between lignin content and breaking strength across different genotypes is illustrated in Figure 3 using linear regression."

Line 210-212: Change "According to the Figure 3., changes in the lignin content at the lower part of the plants exerts greater effect on the changes in the breaking force resistance as opposed to the upper parts." to "As shown in Figure 3, changes in lignin content at the lower part of the plants exert a greater effect on breaking force resistance compared to changes in the upper parts."

Line 212-213: Change "Samples can be grouped according to genotype, but the separation is not so clear based on the stem region studied." to "Samples can be grouped by genotype, but clear separation based on stem region is not evident."

Line 215-217: Change "MANOVA was conducted to determine whether there is a statistically significant interaction between the genotypes used and the stem region on the dependent variables studied." to "A MANOVA was conducted to determine whether there is a statistically significant interaction between the genotypes and stem regions on the dependent variables studied."

Line 246-247: Change "CcoAOMT integrates later in the metabolic process, while CAD serves as the terminal step in forming monolignol units." to "CcoAOMT is integrated later in the metabolic process, while CAD serves as the final step in forming monolignol units."

Line 300-302: Change "While tti mutants exhibited lignin content similar to the control, their breaking strength was notably inferior, potentially due to the elongated structure of the internodes." to "Although the tti mutants exhibited lignin content similar to the control, their breaking strength was notably inferior, potentially due to the elongated structure of the internodes."

Author Response

Dear Reviewer,

I sincerely appreciate the time and effort you have taken to thoroughly review our submitted article. Your thoughtful feedback and valuable suggestions are greatly appreciated, and we are thankful for your insightful remarks.

While the study offers valuable insights into the mechanistic aspects of lignin content and stem rigidity, the discussion on the practical implications for breeding and cultivation is somewhat limited. Expanding on how these findings could be applied in agricultural practices would enhance the study's relevance to the field.

For your suggestion we added a small part related to their appliance in agricultural practices in line 346-349.

Line 18-20: Change "This study investigates the biomechanical properties and lignin content of various Capsicum annuum mutant lines, namely ‘fragile-plant’ (frx), ‘tortuous internodi’ (tti) ‘puffy-structured stem’ (pfi), and, compared to a commercially established variety, 'Garai fehér'." to "This study investigates the biomechanical properties and lignin content of various Capsicum annuum mutant lines—'fragile-plant' (frx), 'tortuous internodi' (tti), and 'puffy-structured stem' (pfi)—in comparison to a commercially established variety, 'Garai fehér'."

As you kindly suggested we corrected this part. Please see in line 19-21.

Line 37-38: Change "Horticulture, alongside crop production and livestock farming, represents one of the most crucial sectors within agriculture." to "Horticulture, alongside crop and livestock production, is one of the most crucial sectors in agriculture."

Following your suggestion we corrected this part. Please see in line 41-42.

Line 70-71: Change "Lignin deficiency can lead to abnormal stem development, highlighting the complex interplay between genetics and biochemical processes in shaping plant architecture." to "Lignin deficiency can cause abnormal stem development, underscoring the complex interplay between genetics and biochemical processes in shaping plant architecture."

Revised in line 75-77.

Line 205-206: Change "The correlation between lignin content and breaking strength in aspect of the various genotypes is illustrated in the following figure using a linear regression." to "The correlation between lignin content and breaking strength across different genotypes is illustrated in Figure 3 using linear regression."

Corrected in line 224-225.

Line 210-212: Change "According to the Figure 3., changes in the lignin content at the lower part of the plants exerts greater effect on the changes in the breaking force resistance as opposed to the upper parts." to "As shown in Figure 3, changes in lignin content at the lower part of the plants exert a greater effect on breaking force resistance compared to changes in the upper parts."

Corrected in line 232-234.

Line 212-213: Change "Samples can be grouped according to genotype, but the separation is not so clear based on the stem region studied." to "Samples can be grouped by genotype, but clear separation based on stem region is not evident."

Revised in line 236-237.

Line 215-217: Change "MANOVA was conducted to determine whether there is a statistically significant interaction between the genotypes used and the stem region on the dependent variables studied." to "A MANOVA was conducted to determine whether there is a statistically significant interaction between the genotypes and stem regions on the dependent variables studied."

Revised in line 240-242.

Line 246-247: Change "CcoAOMT integrates later in the metabolic process, while CAD serves as the terminal step in forming monolignol units." to "CcoAOMT is integrated later in the metabolic process, while CAD serves as the final step in forming monolignol units."

Revised in line 274-275.

Line 300-302: Change "While tti mutants exhibited lignin content similar to the control, their breaking strength was notably inferior, potentially due to the elongated structure of the internodes." to "Although the tti mutants exhibited lignin content similar to the control, their breaking strength was notably inferior, potentially due to the elongated structure of the internodes."

As you kindly suggested we revised in line 330-332.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The English and Format of the revised manuscript have been greatly improved, and the issues I concerned have been clearly addressed, especially in the Section of Materials and Methods. I suggest accept it in present form for publication in this journal.

Author Response

Dear Reviewer,

Thank you kindly for your very useful comments which improved the quality of the manuscript. This way it became clearer and more understandable for everyone.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was considerably improved, however a crucial point is still missing. The absorptivity (e) a very important parameter for the lignin content determination is missing. This will allow future researchers to compare the results using this method. It is not admissible to publish results using this method not stating the value used.

Authors state (133-134) that the value used was from “lignin in N. benthamiana stems”. A more detailed explanation should be given, what kind of lignin, how was it prepared, from which part? Why is this value better than the one from lignin used for the calibration curve?

Has long as this value is provided the manuscript should be acceptable for publication.

Author Response

Dear Reviewer!

We thank you kindly for your comments which improves the quality of the manuscript. As you asked for the absorptivity value, counting the value using the added equation and the path of lenght is 1 cm, ε=62.71 M⁻¹ cm⁻¹.

It might be an english formating and grammatical mistake from our side. In the following sentence we meant that since this is a missing information related to plants from the same family, the value belonging to N. benthamiana is generally used. It might be better that way, that „Currently, no extinction coefficient is available for lignin in pepper stems, so the extinction coefficient for lignin in N. benthamiana stems is generally used instead.”

Both of them were corrected in the manuscript. Please see line 134-136.