Identification and Evaluation of Diterpenoids from Glandular Trichome Secretions of Air/Sun-Cured Tobacco Germplasm Resources

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Manuscript Number: agronomy-2572737

Comments to Authors

 Recommendation:                 

 

The study represented in the article “Identification and Evaluation of Diterpenoids from Glandular Trichome Secretions of Air/Sun-Cured Tobacco Germplasm Resources” was conducted to determine the composition and content of diterpenoids of glandular trichome secretions using ultra-performance liquid chromatography.

 

Although the chosen subject of investigation has its scientific importance, I have several comments that pointed out that the manuscript should be improved:

 Abstract should be corrected:

 

1. Line 20 – please, rewrite “11 chromatographic peaks (Peak1–Peak11) were identified” – in this context, peaks could be observed, detected, corresponding to the identified constituents…
2. The statements in the Abstract are not precisely written - what does variation coefficient mean in the text (Abstract, lines 20,21)?
3. Please, name clearly “the three principal components”, line 25

 

Introduction:

1. Page 2, line 50, the full Latin name of plants should be given (species name, author name and family name). Afterwards, the common name or short name should be used. Please, check throughout the text and make appropriate changes.
2. Page 2, line 59, “Cis” should be italic – please check throughout the manuscript and make appropriate changes
3. Page 2, line 60-61, please rewrite, and make sentence clear
4. Page 2, line 66, please, name the “multiple purposes”. The uninformative statements do not give the scientific soundness to the manuscript, and should be avoided
5. Page 2, lines 66-67 “ (including breeding, local, and introduced resources)” – if these represent the criteria for choosing the germplasms, please point out that
6. Please, rewrite the text page 2, lines 66-69.
7. In Introduction section, the aim and the goals of the performed investigation should be clearly stated, with the stress on the authors’ contribution to the chosen field of investigation.

 

Materials and Methods

1. Table 1 – Page 2, do the mentioned Chinese names of the different varieties of Nicotiana tabacum L. Solanaceae, has some other name, or are they specific for China?
2. Table 1 – please, explain, what type of material used means, what does the “cigar” characterise, and the main differences between material described as “cigar” and “sun-dried”. That should be added in the table, or somewhere in the text, to make sure that those information are shared with the scientific readers
3. Section 2., lines 101-108 – Please, give the concentration of the analysed samples (the date that 10 flowers were taken and put into 50 mL did not revealed the concentration, what is important for further investigation, as all the experiments were based on comparison of quantitative analysis of selected constituents)
4. As the standard curve was mentioned, line 110, please give the name of the used standards, and the concentration  range of standards used for calibration curve

 

Results and Discussion

1. Line 131-133, please, rewrite, it is unclear how the “chromatographic peaks greater than the detection limit (2 μg/mL)” were determined
2. Please, explain: “Data were recorded in spreadsheets using Excel 2010”.
3. Lines 134-136, authors gave the data that identification was performed using GC-MS, but in the Materials and Methods the method was not mentioned
4. The method of the identification should be given (were all quantified compounds identified in comparison to the pure standards, or the MS data were used. If the latter is the case, the appropriate mass fragmentation should be given, as well as the literature data confirming the identification)
5. The quantification of the identified compounds was not explained

The comments were given in the text of comments

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The work is very impressive, and the data is abundant. Nonetheless, some revision need to be done before. Below are my comments.

Line 31

Provide the list of abbreviations.

Line 70

Is it UPLC-MS or UPLC-DAD?

Section 2.4, line 102

Diterpenoids

Talk about how the sample was extracted. This is very important.

 

Subsection Diterpenoid Components of Glandular Trichome Secretion

Line 136

Since you have mentioned GC-MS, you need to GC's chromatographic conditions in the materials and method section.

Line 217

Add reference to that.

Also talk about the future experiments that needs to be done

Minor revision.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments on the revised manuscript agronomy-2572737 “Identification and Evaluation of Diterpenoids from Glandular Trichome Secretions to explore the use of tobacco germplasm resources for multiple purposes”.

 The revised manuscript is slightly improved but still has significant flaws.

The weakness concerning the extraction and analysis procedures, remained.

 The authors replied that “The extraction and analysis procedures on diterpenoid components of plant were established well in our laboratory. Except for reference 14 in manuscript, we added reference 16 and an English reference 17 and more information according to your suggestion in the revised manuscript”.

Although these procedures were well defined in the authors' laboratory, it is important that they are described in detail in the manuscript. In the English summary of ref ex 14 now 15, a different and far more elaborate extraction method is given than that hinted at by the authors in the manuscript. The other two added references (ref 16 is only in Chinese and, ref 17 refers to a further reference) do not help and in any case refer to different analytical situations aimed at other purposes.

As concerns information regarding calibration curves, linearity range, LOD and LOQ, reproducibility requested, the authors replied that they “added a reference and the above information can be referred in reference 15-17 in the revised manuscript”. As mentioned above regarding extraction methods the added references do not help to clarify the required information.

The heart of the experimental work done is precisely the identification and quantitative and semiquantitative analysis of diterpenoids and therefore everything useful to demonstrate the correctness of the procedures chosen and to be able to replicate the data obtained should be detailed in the manuscript.

The description about the identification by GC-MS analysis of the 7 analytes for which semiquantitative analysis was performed (2.5 GC-MS analysis in the Materials and Methods) is missing important information. Is the sample injected in GC-MS the same extract used for analysis in UPLC?  Data on the velocity and scanning range of the masses are missing. How was peak identification done? What library of spectra was used? The data acquisition mode, FULL SCAN (FS) or SELECTED ION MONITORING (SIM) is not indicated. Most importantly, the manuscript does not describe how the GC data were compared with those obtained at UPLC and which allowed the latter to be unequivocally identified.

Regarding field trials, the authors replied that “ the data were the mean of 2020 and 2021”.

Considering that at lines 98-100 the authors report : “ Field trials were conducted at the Cigar Scientific Research and Testing Station of  Sichuan Province and the Luozhuang Experimental Station of Shandong Province in 2020  and 2021” , at line 107 “The field 100 trials were arranged in a randomized block design with three replications” and at line 107 “The 20 flowers were divided into two groups of 10 flowers”, for each tobacco, is the data the mean of 24 extractions and determinations (2 locations x 2 years x 3 replications x 2 groups) ?.

The authors replied that “The same planting condition and the average values of the two years can eliminate variations caused by environmental factors”. In my opinion this sentence is inaccurate, it is not average values that eliminate variation, but average values can be presented in the absence of main effects of location and year and interaction with genotype.

 Other Comments

Line 21 : " ....where detected by UPLC identified...." : “identified” should be delete.

Line 21 : reference 2 is the same as 5.

Line 66: oriental tobaccos are classified as sun cured.

Line 75: reference/s is missing.

Line 75-77 : the sentence " The lack of systematic identification of diterpenoid compounds such as CBT-diol, cis -abieol and CBT-ol in sun/air-cured tobacco germplasm resources. " is incomplete, the verb is missing.

Line 95-96: some changes were made to Table 1 (in the column “type” cigar was replaced with air-cured) and a note was added. It is always appropriate to highlight the changes. 

However, the note specifies “The air-cured tobacco germplasm resources here are cigar tobacco. X78 germplasm resource is oriental tobacco”.

What type of tobacco are the local ones? Non-Oriental sun-cured tobacco and therefore similar to what type of tobacco?  In my opinion more than the indication of the curing process in the table should be stated the type of tobacco: Burley, Maryland, Bright, Kentucky, Oriental  (Perustitza, Herzegovina, Xanthi etc.) etc..  This is to associate the results of the diterpene content with the type of tobacco (burley tobaccos are also air-cured , and cigar tobaccos are also Kentucky, fire-cured).

In my opinion “air/sun” cured remains uninformative. “

 Line 115: the type of filter is still missing (Nylon, Cellulose acetate…?)

 Line 126: in "The detection column ...." detection should be delete.

Line 164-165: the sentence " Solvent peaks were removed, and the common chromatographic peaks greater than limit of quantitation the detection limit (2 μg/mL) were labeled Peak1–Peak11 from left to right (Figure 2)" is incomplete.

Line 175: in fig.3 retention times should also be included for peaks in the chromatogram referring to germplasm. Furthermore, the chromatogram referring to CBT-diol appears to have been obtained by injecting a standard containing both isomers; it should be reported how these isomers were quantified.

Line 188: in Table 3 the unit of measurement must be completed with dry weight or fresh weight.

 Line 243: in Table 4 the unit of measurement must be completed with dry weight or fresh weight. The names of the terpenoids should be added to the legend in Table 4.

 Line 284: in Table 8 the unit of measurement must be completed with dry weight or fresh weight.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments on the revised manuscript agronomy-2572737

 

Although improved, still the manuscripts need substantial corrections.

Mostly, the answers were pointed out to the reviewer.

 

The weakness regarding the extraction and analysis procedures, performed identification and quantification, on which the rest of the experiments were based, remained.

The identification and quantification of the main groups of secondary metabolites were not presented clearly. The authors did provide the data regarding the standard used, but the method of extraction was not given, the given column data in GC/MS methodology refers to catalogue number, which was not requested, and the equation is not precisely given, as the explanations are not scientifically given – “ … where Cn is the content of the peak of j and Cβ-CBT-diol is the content of β-CBT-diol; Sn 141 indicates the peak area of n (n = 1, 2..., 11) and Sβ-CBT-diol indicates the peak area of β-CBT- 142 diol” page  5, line 135-143 (the terminology is not acceptable), the authors did not provide the source of the standard of CBT-ol and CBT-diols, how they were dissolved and the used concentration for identification and further for quantification. It refers to the rest of the mentioned main compounds used to perform further analysis. Considering that the analytical part of the submitted article represents the base for performed data analysis, the UPLC and GC/MS analysis should be given in detail, which was not corrected in the revised version.

Table 2, what does the legend for Table 2 mean: “ Table 2. Basic data characteristics of 11 chromatographic peaks of glandular trichome secretions”

 

Regarding the explanation of “variation coefficient (Abstract, lines 20,21)”, the terminology remained incorrect. Authors should correct it into “coefficient of variation”

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have responded to my comments. 

Minor editing 

Author Response

Thank you once again for your valuable guidance and support.