Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production
Round 1
Reviewer 1 Report
In overall, the manuscript is well elaborated and presents novel results. The research presents quite an extensive study of the interconnection of the effect of putrescine on betulin production and associated rise of NO and NH4+ content in birch cells. The introduction is objective and brief. Authors used reasonable and scientifically valuable methods to explain the role of NO in betulin production induced by putrescine. Results are clearly described and adequately discussed. The English of presented manuscript is of a good quality and understandable.
I have few notes to the authors:
Introduction chapter: For the molecules such as nitric oxide (NO), or hydrogen peroxide (H2O2) are given full names when first mentioned, unlike for the ammonium cation (NH4+) where only chemical formula is given.
Row 37 – it seems that the first sentence of second paragraph miss the predicate.
Row 54 – there is a typing error in lupeol synthase
Results chapter: the results are expressed mostly using a bar chart, except one case where a plot chart is used (Figure 2). All charts (except Figure 6) represents effect of Putrescine on different factors in birch cells. The charts should be therefore unified for easier comparability.
Charts in figure 5 deserve some improvement. For example increase size and make Y caption in one line in all charts so the charts would be aligned.
Figures should be self-explanatory, so at least the worse detectable abbreviations should be explained in figure caption. For example NS and NCL in Figure 6, or LUS (although generally used for lupeol synthase, it is not so common).
Materials and Methods: A space between number and unit is missing in few cases and should be added.
The chapter 4.3 describes poorly the extraction of betulin and no purification procedure is mentioned. The description in the reference article (Fan et al, 2014) is also quite brief. The true is, that the betulin estimation is very simple, still I have few questions. Did you performed extraction from fresh birch cells or dried? What was the proportion of biomass and solvent? Did you apply crude ethanol extract to HPLC or did you use any filtration or purification? The betulin content in the Results chapter is related to DW of crude extract or dry biomass (cells)? In case it is applied to the extract, the extract yield should be mentioned, since it may differ according to the particular treatment. Also the ratio of fresh and dry mass may differ in response to applied elicitors/stimulators or inhibitors. The length of HPLC analysis run time should be mentioned as well.
The chapter 4.4. – did you apply any modifications to the manufacturer´s instructions of the assay kits?
The chapter 4.6 – the sample preparation (protein extraction and protein quantification) applied to assay kits is not described. At least briefly what buffer was used and what method of protein quantification was used for determination of GS, GDH and GOGAT activity. What was the protein concentration applied to assay kit (if adjusted).
The assay buffer used for determination of NR activity is not specified.
The chapter 4.7 – the RNA isolation and quantitative real-time PCR analysis is also described very briefly and the reference given (18) does not contain results based on this method at all. I suggest to add relevant references.
Author Response
Introduction chapter: For the molecules such as nitric oxide (NO), or hydrogen peroxide (H2O2) are given full names when first mentioned, unlike for the ammonium cation (NH4+) where only chemical formula is given.
Reply: ammonium catio(NH4+) was added in row 42.
Row 37 – it seems that the first sentence of second paragraph miss the predicate.
Reply: NO was considered a bioactive signaling groundbreaking described in plants
Row 54 – there is a typing error in lupeol synthase
Reply: It has been modified
Results chapter: the results are expressed mostly using a bar chart, except one case where a plot chart is used (Figure 2). All charts (except Figure 6) represents effect of Putrescine on different factors in birch cells. The charts should be therefore unified for easier comparability.
Reply: It has been modified
Charts in figure 5 deserve some improvement. For example increase size and make Y caption in one line in all charts so the charts would be aligned.
Reply: It has been modified
Figures should be self-explanatory, so at least the worse detectable abbreviations should be explained in figure caption. For example NS and NCL in Figure 6, or LUS (although generally used for lupeol synthase, it is not so common).
Reply: we explained it in paper. In addition, the abbreviations of LUS is used for lupeol synthase in the published paper, such as Transcriptome analysis identifies key genes involved in the regulation of epidermal lupeol biosynthesis in Ricinus communis (Industrial Crops and Products , 2020, 113100)
Materials and Methods: A space between number and unit is missing in few cases and should be added.
Reply: It has been modified.
The chapter 4.3 describes poorly the extraction of betulin and no purification procedure is mentioned. The description in the reference article (Fan et al, 2014) is also quite brief. The true is, that the betulin estimation is very simple, still I have few questions. Did you performed extraction from fresh birch cells or dried? What was the proportion of biomass and solvent? Did you apply crude ethanol extract to HPLC or did you use any filtration or purification? The betulin content in the Results chapter is related to DW of crude extract or dry biomass (cells)? In case it is applied to the extract, the extract yield should be mentioned, since it may differ according to the particular treatment. Also the ratio of fresh and dry mass may differ in response to applied elicitors/stimulators or inhibitors. The length of HPLC analysis run time should be mentioned as well.
Reply: 0.50 g powder of dried suspension cells was soaked in 20 mL hydrochloric acid:ethanol solution (2:8 by volume). The solution was refluxed for 3 h in a water bath at 90 °C. The filtrate was extracted three times with 15 mL of ether. The combined extracts were evaporated to dryness at 40 °C and redissolved in 2 mL ethanol for HPLC analysis.
The length of HPLC analysis run time was 13.5 min.
The chapter 4.4. – did you apply any modifications to the manufacturer´s instructions of the assay kits?
Reply: no
The chapter 4.6 – the sample preparation (protein extraction and protein quantification) applied to assay kits is not described. At least briefly what buffer was used and what method of protein quantification was used for determination of GS, GDH and GOGAT activity. What was the protein concentration applied to assay kit (if adjusted).
Reply: we added it.
The assay buffer used for determination of NR activity is not specified.
Reply: we added it.
The chapter 4.7 – the RNA isolation and quantitative real-time PCR analysis is also described very briefly and the reference given (18) does not contain results based on this method at all. I suggest to add relevant references.
Reply: relevant references was reference 33.
Reviewer 2 Report
The present research by Guizhi Fan and colleagues provides relevant pieces of information for potential improvement of biotechnological production of betulin.
I feel, however, that the ms needs considerable revamp before publication:
- In general, help the reader by providing an introductory sentence, at the beginning of every results section, that motivates the experiments that are presented
- Figures need a legend. Provide all related information (time of the treatments, volumes, replicates, statistical tests, meaning of acronyms, etc)
- Results section 2.5 – please develop and explain more in detail.
Minor comments:
Line 67 – more than inhibitory, it looks like no significant effect, or slightly increased betulin production at 12 hours.
Line 88 – briefly explain what is and why to measure NO fluorescence, to readers not familiar with it.
Line 90 – Sorry, I cannot see the maximum NO fluorescence at 12h in any of the treatments according to Fig2B
Figure 2 – what is C1-to-C6??
Line 96 – I can see that both treatments NOS enzyme activity peaked only at 3h of the two treatments
Figure 6 title does not correspond with what is shown.
Line 211 – which confocal system?
Line 235 – please, provide a reference for the CTAB-based protocol
Author Response
- In general, help the reader by providing an introductory sentence, at the beginning of every results section, that motivates the experiments that are presented
- Reply: we added it. such as Betulin is lupane-type triterpenes, one of its synthetic key enzymes is lupeol synthase (LUS); NR and NOS are the two NO-generating key enzymes in plant.
- Figures need a legend. Provide all related information (time of the treatments, volumes, replicates, statistical tests, meaning of acronyms, etc)
- Reply: we added it.
- Results section 2.5 – please develop and explain more in detail.
- Reply: we added it.
Minor comments:
Line 67 – more than inhibitory, it looks like no significant effect, or slightly increased betulin production at 12 hours.
Reply: It has been modified.
Line 88 – briefly explain what is and why to measure NO fluorescence, to readers not familiar with it.
Reply: we added reference 30 to explain it.
Line 90 – Sorry, I cannot see the maximum NO fluorescence at 12h in any of the treatments according to Fig2B
Reply: it was the average fluorescence intensity of 10~15 pictures.
Figure 2 – what is C1-to-C6??
Reply: we added it.
Line 96 – I can see that both treatments NOS enzyme activity peaked only at 3h of the two treatments
Reply: NOS enzyme activity peaked at 3h after 0.1mmol·L-1 and 1mmol·L-1 Put treatment.
Figure 6 title does not correspond with what is shown.
Reply: It has been modified.
Line 211 – which confocal system?
Reply: we added it.
Line 235 – please, provide a reference for the CTAB-based protocol
Reply: we added it.
Reviewer 3 Report
A comprehensive and interesting study with a significant impact on the efficient production of secondary metabolites.
The manuscript requires more formal adjustments and additions to the methodology of the experiments. Some suggestions for editing are marked directly in the text of the manuscript.
The figure 1, 3 and 5 should be enlarged.
Relative expression of LUS gene in fig. 1 and 4 should include the units (delta delta Ct).
The figure 2 is insufficiently described. Explanations of individual designations are missing (2A, 2B, C1-C6).
There is missing reference of Nagata-Takebe medium.
What is the origin of the suspension cells cultures. This information should be included in methodology.
Reference 21 in the reference list is not correct.
Comments for author File: 
Comments.pdf
Author Response
he manuscript requires more formal adjustments and additions to the methodology of the experiments. Some suggestions for editing are marked directly in the text of the manuscript.
The figure 1, 3 and 5 should be enlarged.
Reply: It has been modified.
Relative expression of LUS gene in fig. 1 and 4 should include the units (delta delta Ct).
Reply: Relative expression was the ratio of put treatment/control.
The figure 2 is insufficiently described. Explanations of individual designations are missing (2A, 2B, C1-C6).
Reply: we added it.
There is missing reference of Nagata-Takebe medium.
Reply: we added it.
What is the origin of the suspension cells cultures. This information should be included in methodology.
Reply: we added it.
Reference 21 in the reference list is not correct.
Reply: It has been modified.
Round 2
Reviewer 2 Report
I acknowledge that the authors have improved the quality of the manuscript´s presentation. However, the present revised version is still overlooking one of my main concerns. All result sections should begin with an introductory sentence/paragraph that motivates the experiments that are presented. I cannot recommend the editor to accept this manuscript for publication until it meets such an important (and minimum) requirement of scientific style.
Specific comments:
Line 67 – was decreased with respect to what? It is not significantly different from control. This sentence is still confusing.
Line 101 – B panel is NOS, not NR.
Figure 4 and 6 – I believe the legends remains insufficiently self-explanatory. The treatments should be explained shortly here.
Author Response
I acknowledge that the authors have improved the quality of the manuscript´s presentation. However, the present revised version is still overlooking one of my main concerns. All result sections should begin with an introductory sentence/paragraph that motivates the experiments that are presented. I cannot recommend the editor to accept this manuscript for publication until it meets such an important (and minimum) requirement of scientific style.
Reply: It has been modified
Specific comments:
Line 67 – was decreased with respect to what? It is not significantly different from control. This sentence is still confusing.
Reply: It has been modified
Line 101 – B panel is NOS, not NR.
Reply: It has been modified
Figure 4 and 6 – I believe the legends remains insufficiently self-explanatory. The treatments should be explained shortly here.
Reply: It has been modified
