Anthocyanin Accumulation and Differential Expression of the Biosynthetic Genes Result in a Discrepancy in the Red Color of Herbaceous Peony (Paeonia lactiflora Pall.) Flowers

Round 1

Reviewer 1 Report

I have thoroughly reviewed the ms entitled " Anthocyanin accumulation and differential expression of the biosynthetic genes result in a discrepancy in the red color of the herbaceous peony (Paeonia lactiflora Pall.) flowers", the authors have developed a study of the difference in floral morphology, biosystematic expression of pigments involved in floral color, underlying gene expression and sugar synthesis, between two cultivars of Paeonia lactiflora. The work is very descriptive and makes comparisons with previous information on cultivars or related species, highlighting the discrepancies observed with respect to the literature. The methodology is correct and the text is concise and straightforward with an impeccable use of English. I therefore recommend this manuscript for publication in Horticulturae. Only, I would strongly suggest that, for readers unfamiliar with floral pigment biosynthetic pathways, I recommend including a figure with a schematic pathway showing, at least, at which point in the pathway each of the genes analyzed in the study acts. This would simplify the understanding of the work and would allow a clearer visualization of where in the pathway there are differences between the two cultivars. I also recommend including some more references on biosynthetic pathways in floral color from paper developed around the world. It is not only in China where this kind of studies are carried out. In addition, I suggest some minor changes below:

 

• In the title “Paeonia lactiflora” must be in italics
• I recommend including the objectives of the work more explicitly at the end of the introduction.
• In line 116: what do the authors mean by “cDNAs”? this can be very generic. I recommend being more precise in this sentence.
• In line 122: for “…was determined according to the method of paper weighing.” include a reference.
• In line 124: write “Weight of Fresh flowers” instead of “Fresh weights of the flowers”.
• In line 176: Can the authors clarify why they use PlPAL gene as control? Are they based on previous work? Why?
• In lines 194-195: for “The light response 194 model parameters were estimate using SPSS16.0”, authors should specify how this analysis was performed: which model, Link function, factors (fixed or random), normality of the data.....
• Finally, in conclusion section: Although the conclusion is correct, I think the authors could use the publication in Horticulturae to highlight the importance of such studies in commercial strategies. Might the synthesis of different petal pigments have economic consequences?

Author Response

Reviewer’s comments and suggestions 1:

1. Reviewer #1: I have thoroughly reviewed the ms entitled "Anthocyanin accumulation and differential expression of the biosynthetic genes result in a discrepancy in the red color of the herbaceous peony (Paeonia lactifloraPall.) flowers", the authors have developed a study of the difference in floral morphology, biosystemtic expression of pigments involved in floral color, underlying gene expression and sugar synthesis, between two cultivars of Paeonia lactiflora. The work is very descriptive and makes comparisons with previous information on cultivars or related species, highlighting the discrepancies observed with respect to the literature. The methodology is correct and the text is concise and straightforward with an impeccable use of English. I therefore recommend this manuscript for publication in Horticulturae. Only, I would strongly suggest that, for readers unfamiliar with floral pigment biosynthetic pathways, I recommend including a figure with a schematic pathway showing, at least, at which point in the pathway each of the genes analyzed in the study acts. This would simplify the understanding of the work and would allow a clearer visualization of where in the pathway there are differences between the two cultivars. I also recommend including some more references on biosynthetic pathways in floral color from paper developed around the world. It is not only in China where this kind of studies are carried out.

Answer: Thank you for your recognition of this study very much. According to your comments and suggestions, the anthocyanin biosynthesis pathway in plants has been added in Figure S1. And we have added some more references on biosynthetic pathways in color around the world. Please check below. Thank you for your comments very much.

 

Figure S1 The schematic diagram of anthocyanin biosynthesis pathway in plants (Frontiers in Plant Science, 2015, 6: 261). PAL, phenylalanine ammonia lyase gene; C4H, cinnamate-4-hydroxylase gene; 4CL, 4-coumarate: CoA ligase gene; CHS, chalcone synthase gene; CHI, chalcone isomerase gene; F3H, flavanone 3-hydroxylase gene; F3'H, flavonoid 3'-hydroxylase gene; F3',5'H. flavonoid 3',5'-hydroxylase gene; DFR, dihydroflavonol 4-reductase gene; ANS, anthocyanidin synthase gene; UFGT, UDP-glucose: flavonoid glucosyltransferase gene; MT, methyl transferase gene.

Filyushin, M.A.; Dzhos, E.A.; Shchennikova, A.V.; Kochieva, E.Z. Dependence of pepper fruit colour on basic pigments ratio and expression pattern of carotenoid and anthocyanin biosynthesis genes. Russ. J. Plant Physl. 2020, 67, 1054-1062. (doi: 10.1134/S1021443720050040)

Hossain, M.R.; Kim, H.T.; Shanmugam, A.; Nath, U.K.; Goswami, G.; Song, J.Y.; Park, J.I.; Nou, I.S. Expression profiling of regulatory and biosynthetic genes in contrastingly anthocyanin rich strawberry (Fragaria x ananassa) cultivars reveals key genetic determinants of fruit color. Int. J. Mol. Sci. 2018, 19, 656. (doi: 10.3390/ijms19030656)

Karanjalker, G.R.; Ravishankar, K.V.; Shivashankara, K.S.; Dinesh, M.R.; Roy, T.K.; Rao, D.V.S. A study on the expression of genes involved in carotenoids and anthocyanins during ripening in fruit peel of green, yellow, and red colored mango cultivars. Appl. Biochem. Biotech. 2018, 184, 140-154. (doi: 10.1007/s12010-017-2529-x)

Maritim, T.K.; Masand, M.; Seth, R.; Sharma, R.K. Transcriptional analysis reveals key insights into seasonal induced anthocyanin degradation and leaf color transition in purple tea (Camellia sinensis (L.) O. Kuntze). Sci. Rep. 2021, 11, 1244. (doi: 10.1038/s41598-020-80437-4)

2. Reviewer #1: In the title “Paeonia lactiflora” must be in italics.

Answer: We are very sorry for our carelessness. And we have revised “Paeonia lactiflora” to be in italics. Thank you for your comments very much.

3. Reviewer #1: I recommend including the objectives of the work more explicitly at the end of the introduction.

Answer: Thank you for your comments very much. “In order to elucidate how anthocyanins affect the different color depths of red flowers and which gene is dominant in the process, a red cultivar of P. lactiflora (‘Dafugui’) and a pink cultivar (‘Qingwen’) (Figure 1) were selected for studying the discrepancy in the red color of the flowers in this study.” has been added in lines 84-88. Thanks again.

4. Reviewer #1: In line 116: what do the authors mean by “cDNAs”? this can be very generic. I recommend being more precise in this sentence.

Answer: We are very sorry for our carelessness. cDNAs are obtained by reverse transcription of RNA, which will be used for subsequent gene expression. Because “synthesizing cDNAs” is easy to misunderstand here, we has been deleted. Thank you for your comments very much.

5. Reviewer #1: In line 122: for “…was determined according to the method of paper weighing.” include a reference.

Answer: We are very sorry for our carelessness. Because this method is often used in China, we did not add it. And the reference “Zhao, D.Q., Sheng, Y.L., Zhou, C.H. Method of non-destructive measurement research on leaf area and fruit fresh weight of Ginkgo biloba. North. Hortic. 2010, 7, 28-30. (doi: 10.11937/bfyy.201007009)” has been added. Thank you for your comments very much.

6. Reviewer #1: In line 124: write “Weight of Fresh flowers” instead of “Fresh weights of the flowers”.

Answer: Thank you for your comments very much. We have revised “Fresh weights of the flowers” to “Weight of Fresh flowers”. Thanks again.

7. Reviewer #1: In line 176: Can the authors clarify why they use PlPALgene as control? Are they based on previous work? Why?

Answer: Thank you for your comments very much. “the expression level of PlPAL in S1 of ‘Dafugui’ was used as the control” means that its expression level is 1.0 to adjusted to the expression levels of other genes, so that the relative expression levels of different genes can be compared. And this method has been used in our previous studies (Biochemical and Biophysical Research Communications, 2015, 459: 450e456; Plant Physiology and Biochemistry, 2016, 102: 97-106). Thanks again.

8. Reviewer #1: In lines 194-195: for “The light response model parameters were estimate using SPSS16.0”, authors should specify how this analysis was performed: which model, Link function, factors (fixed or random), normality of the data.....

Answer: Thank you for your comments very much. According to the comments of two other reviewers, “3.5. Soluble sugar content and photosynthetic characteristics” should be deleted because it is not related to pigmentation in this manuscript. Therefore, we have deleted the corresponding Materials and Methods section. Thanks again.

9. Reviewer #1: Finally, in conclusion section: Although the conclusion is correct, I think the authors could use the publication in Horticulturae to highlight the importance of such studies in commercial strategies. Might the synthesis of different petal pigments have economic consequences?

Answer: Thank you for your comments very much. We also want to publish this manuscript in Horticulturae to highlight the importance of such studies in commercial strategies. In addition, the sentence “This study can serve as a theoretical basis for breeding more red flowers using genetic engineering techniques to cater to the preferences of consumers.” has been added in lines 358-360.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript ‘Anthocyanin accumulation and differential expression of the biosynthetic genes result in a discrepancy in the red color of the herbaceous peony (Paeonia lactiflora Pall.)’ by Yanqing et al. describes the anthocyanin accumulation and coloring mechanism of red vs pink flowered cultivar. Author point at the differential gene expression related in anthocyanin biosynthesis led to discrepancy in coloration of peony flowers, despite of a same composition of anthocyanin between two cultivars. Authors claim that their observations indicate a soluble sugar was negative correlation with anthocyanin content of peony flower.

Firstly, there is a serious problem with the interpretation of the study. Which is the crucial factor for flower color difference between composition and content of anthocyanin? Authors need to carefully review factors related to differential pigmentation and describe them in deep discussion. Previous studies reported that biosynthesis of flavonoids including flavonols and flavanones can effect on anthocyanin biosynthesis. Based on these results, authors should indicate the evidence of the different transcript level of genes related in flavonols and flavones biosynthesis in flower. Based on gene expression analysis, authors describe the effect on the transcript level in the text.

 

Second, there is containing many off-topic for this study. Authors need to rearrange and rewrite the manuscript.

Any abbreviation must be associated with the full name at the first mention in the manuscript to allow the reader to follow up because not all the readers are familiar with the abbreviated terminology.

In the results part,

I do not know the relationship between morphological parameter and flower color. If need it, it should be transferred to the supplement data. Please provide a reason why this text should be mentioned in the manuscript.

There is not mentioned the relevance between flower color and flower diameter. It is better to omit the off-topic in the manuscript for providing a clear meaning for this study.

The result on flower colorimetric should change into a table format with a clear meaning. The value of H0 was not consistence with the interpretation in the manuscript.

In the 3.3 part,

According to the data shown in Fig. 4, the compositions of anthocyanins, flavones, and flavonols were similar levels in two varieties, but the content of those was very different. Therefore, it is required to mention the content of the main difference in detail and scientifically analyze the difference in flower color according to the difference.

In the 3.4 part,

Please check the expression of FLS/FNS gene in order to confirm the result of high flavonol and flavonone content in pink flower. Also, please suggest an interpretation of the expression results of major genes affecting the flower color of red and pink flowers.

Please write the name of gene in result of Figure 6 and the developmental stage of two cultivars on the X-axis.

Please suggest whether the results presented in 3.5 are related to pigmentation in this paper.

Author Response

Reviewer’s comments and suggestions 2:

1. Reviewer #2:The manuscript ‘Anthocyanin accumulation and differential expression of the biosynthetic genes result in a discrepancy in the red color of the herbaceous peony (Paeonia lactifloraPall.)’ by Yanqing et al. describes the anthocyanin accumulation and coloring mechanism of red vs pink flowered cultivar. Author point at the differential gene expression related in anthocyanin biosynthesis led to discrepancy in coloration of peony flowers, despite of a same composition of anthocyanin between two cultivars. Authors claim that their observations indicate a soluble sugar was negative correlation with anthocyanin content of peony flower. Firstly, there is a serious problem with the interpretation of the study. Which is the crucial factor for flower color difference between composition and content of anthocyanin? Authors need to carefully review factors related to differential pigmentation and describe them in deep discussion. Previous studies reported that biosynthesis of flavonoids including flavonols and flavanones can effect on anthocyanin biosynthesis. Based on these results, authors should indicate the evidence of the different transcript level of genes related in flavonols and flavones biosynthesis in flower. Based on gene expression analysis, authors describe the effect on the transcript level in the text.

Answer: Thank you for your comments very much. According to the results, we believe that the content of anthocyanins is the crucial factor affecting flower color in these two cultivars. And according to your comments, we have revised the discussion section, please check it. Thanks again.

2. 2. Reviewer #2:Second, there is containing many off-topic for this study. Authors need to rearrange and rewrite the manuscript.

Answer: Thank you for your comments very much. According to your comments and suggestions, “3.5. Soluble sugar content and photosynthetic characteristics” should be deleted, and shorten this manuscript. Please check it. Thanks again.

3. 3. Reviewer #2:Any abbreviation must be associated with the full name at the first mention in the manuscript to allow the reader to follow up because not all the readers are familiar with the abbreviated terminology.

Answer: We are very sorry for our carelessness. We have explained all abbreviations at the first mention in the manuscript, please check below.

(1) in line 60, “HPLC” has been revised to “high-performance liquid chromatography (HPLC)”.

(2) in line 25, “PlANS and PlUF3GT” has been revised to “thocyanidin synthase gene (PlANS) and UDP-Glucose: flavonoid 3-O-glucosyltransferase gene (PlUF3GT)”.

4. 4. Reviewer #2:I do not know the relationship between morphological parameter and flower color. If need it, it should be transferred to the supplement data. Please provide a reason why this text should be mentioned in the manuscript.

Answer: Thank you for your comments very much. The morphological parameters have been transferred to the supplement data. Because two different cultivars are used in this study, comparison of plant morphological indicators can help to differentiate cultivars. Thanks again.

5. 5. Reviewer #2:There is not mentioned the relevance between flower color and flower diameter. It is better to omit the off-topic in the manuscript for providing a clear meaning for this study.

Answer: Thank you for your comments very much. There is no correlation between flower color and flower diameter. Because two different cultivars are used in this study, comparison of flower diameter can help to differentiate cultivars. Thanks again.

6. 6. Reviewer #2:The result on flower colorimetric should change into a table format with a clear meaning. The value of H0 was not consistence with the interpretation in the manuscript.

Answer: Thank you for your comments very much. We have checked the manuscript, the value of H^o was consistence with the interpretation, and a bar chart is more intuitive. Thanks again.

7. 7. Reviewer #2:In the 3.3 part,according to the data shown in Fig. 4, the compositions of anthocyanins, flavones, and flavonols were similar levels in two varieties, but the content of those was very different. Therefore, it is required to mention the content of the main difference in detail and scientifically analyze the difference in flower color according to the difference.

Answer: We are very sorry for our carelessness. Because the compositions are the same, we did not count the specific content of each composition, but it can be seen from the ordinate in Figure 4 that the area of each peak is different. Thank you for your comments very much, and we'll take your advice for future research. Thanks again.

8. 8. Reviewer #2:In the 3.4 part,please check the expression of FLS/FNS gene in order to confirm the result of high flavonol and flavonone content in pink flower. Also, please suggest an interpretation of the expression results of major genes affecting the flower color of red and pink flowers.

Answer: Thank you for your comments very much. In herbaceous peony, the schematic diagram of anthocyanin biosynthesis pathway was figured in previous study (Molecular Biology Reports, 2012, 39: 11263-11275; Electronic Journal of Biotechnology, 2012, 15: 6), and no FLS/FNS gene was found.

 

   In discussion, there was an interpretation of the expression results of major genes affecting the flower color of red and pink flowers. “Whereas in ‘Qingwen’, the upstream substances was insufficient because of the low expression levels of early genes PlPAL and PlF3'H. And the formation of anthocyanins was inhibited because of the low expression levels of the late genes PlANS and PlUF3GT, which resulted in a light pink color of petals.”. Thanks again.

9. 9. Reviewer #2:Please write the name of gene in result of Figure 6 and the developmental stage of two cultivars on the X-axis.

Answer: Thank you for your comments very much. We have revised the legend of Figure 6 to “Figure 6. Expression analysis of the anthocyanin biosynthetic genes in the flower of P. lactiflora cv. ‘Dafugui’ and ‘Qingwen’. PlPAL: phenylalanine ammonialyase gene; PlC4H: cinnamate 4-hydroxylase gene; PlCHS: chalcone synthase gene; PlCHI: chalcone isomerase gene; PlF3H: flavanone 3-hydroxylase gene; PlF3'H: flavonoid 3'-hydroxylase gene; PlDFR: dihydroflavonol reductase gene; PlANS: anthocyanidin synthase gene; PlUF3GT: UDP-Glucose: flavonoid 3-O-glucosyltransferase gene; PlUF5GT: UDP-Glucose: flavonoid 5-O-glucosyltransferase gene. For stage designations, see Figure 2. The values represent the mean ± SDs, and different letters indicate significant differences (P < 0.05).”. Thanks again.

10. 10. Reviewer #2:Please suggest whether the results presented in 3.5 are related to pigmentation in this paper.

Answer: Thank you for your comments very much. According to your comments and suggestions, “3.5. Soluble sugar content and photosynthetic characteristics” should be deleted, and shorten this manuscript. Please check it. Thanks again.

Author Response File: Author Response.docx

Reviewer 3 Report

The reviewed paper is dedicated to the comparison of two peony cultivars with respect to several morphological and physiological characteristics, neither more nor less. The obtained results are of certain interest to readers, especially in the part connected with revealing the mechanisms underlying color differences. I like how quantitative data are presented (although there may be some additional changes).
However, I recommend authors to avoid over-generalizations and overestimations of their experimental findings. For example, there are two parameters which differ between two studied cultivars, viz. content of sugars and content of anthocyanins. As these cultivars were not isogenous lines, they may differ in very many aspects, but it does not mean that all these differences (even if statistically correlated) have a causative interaction. I strongly recommend authors either to shorten their paper (removing everything which is not obviously related to flower color) or to rephrase most of their conclusions to make them more accurate and less shaky.
A serious language/style correction is needed. One of the most constant flaws is connected with the exact role of different factors. Genes encode enzymes, which, in their turn, can catalyze certain reactions. Authors are recommended to elaborate this throughout the whole text.
Additional comments and suggestions are available in the manuscript file (see attached).
I would like to encourage authors to provide an improved version of their paper, which, I am sure, can be recommended for publication, if all (or most of) recommendations are taken into account.

Comments for author File: Comments.pdf

Author Response

Reviewer’s comments and suggestions 3:

1. Reviewer #3: The reviewed paper is dedicated to the comparison of two peony cultivars with respect to several morphological and physiological characteristics, neither more nor less. The obtained results are of certain interest to readers, especially in the part connected with revealing the mechanisms underlying color differences. I like how quantitative data are presented (although there may be some additional changes).However, I recommend authors to avoid over-generalizations and overestimations of their experimental findings. For example, there are two parameters which differ between two studied cultivars, viz. content of sugars and content of anthocyanins. As these cultivars were not isogenous lines, they may differ in very many aspects, but it does not mean that all these differences (even if statistically correlated) have a causative interaction. I strongly recommend authors either to shorten their paper (removing everything which is not obviously related to flower color) or to rephrase most of their conclusions to make them more accurate and less shaky.

Answer: Thank you for your comments very much. According to your comments and suggestions, “3.5. Soluble sugar content and photosynthetic characteristics” should be deleted, and shorten this manuscript. Please check it. Thanks again.

2. 2. Reviewer #3: A serious language/style correction is needed. One of the most constant flaws is connected with the exact role of different factors. Genes encodeenzymes, which, in their turn, can catalyzecertain reactions. Authors are recommended to elaborate this throughout the whole text.

Answer: Thank you for your comments very much. This manuscript has been copyedited by a professional English editing service. As follows:

 

 

3. 3. Reviewer #3:In line 4, italicize “Paeonia lactiflora”.

Answer: We are very sorry for our carelessness. “Paeonia lactiflora” has been italicized. Thank you for your comments very much.

4. 4. Reviewer #3:In line 13, “constitute”should be revised to “constitutes”.

Answer: We are very sorry for our carelessness. This sentence has been revised to “Herbaceous peony (Paeonia lactiflora Pall.) is an ornamental plant having huge potential in the international flower market.”. Thank you for your comments very much.

5. 5. Reviewer #3:In line 14,what “other flowers” are you writing about?

Answer: Thank you for your comments very much. “other flowers” means the flowers of most other ornamental plants. “Like most of the other flowers” has been revised to “Like the flowers of most other ornamental plants”. Thanks again.

6. 6. Reviewer #3:In lines 23-24,I recommend to rephrase: genes encoding phenylalanine ammonialyse (PlPAL) and...

Answer: Thank you for your comments very much. “the early genes phenylalanine ammonialyase gene (PlPAL) and flavonoid 3'-hydroxylase (PlF3'H) gene” has been revised to “the early genes encoding phenylalanine ammonialyase (PlPAL) and flavonoid 3'-hydroxylase (PlF3'H)”. Thanks again.

7. 7. Reviewer #3:In lines 24-25,if you use italics, it means genes. Genes themselves cannot 'catalyze' anything. Therefore it is more correct to write about them 'controlling', 'governing' or 'encoding enzymes which catalyze'.

Answer: Thank you for your comments very much. “Moreover, the catalysis of PlANS and PlUF3GT was found to transform these substances into colored anthocyanins resulting in more redness in ‘Dafugui’ than ‘Qingwen’.” has been revised to “Moreover, the anthocyanidin synthase gene (PlANS) and UDP-Glucose: flavonoid 3-O-glucosyltransferase gene (PlUF3GT) encoding enzymes which catalyze these upstream substances into colored anthocyanins resulting in more redness in ‘Dafugui’ than ‘Qingwen’.” Thanks again.

8. 8. Reviewer #3:In line 35(Herbaceous peony), maybe it is worth mentioning once that it belongs to Paeoniaceae. Thanks again.

Answer: Thank you for your comments very much. “Herbaceous peony (Paeonia lactiflora Pall.) is a herbaceous perennial plant” has been revised to “Herbaceous peony (Paeonia lactiflora Pall.), belonging to Paeoniaceae, is a herbaceous perennial plant”. Thanks again.

9. Reviewer #3: In line 38, verbs like this “reported” inevitably require citations.

Answer: Thank you for your comments very much. The reference “Wang, J.G.; Zhang, Z.S. Herbaceous peonies of China. China Forestry Publishing House, Beijing, 2005.” has been cited. Thanks again.

10. Reviewer #3: In lines 39-41,I think, 'eight groups' is enough to avoid saying 'group' eight times afterwards.

Answer: Thank you for your comments very much. “Based on the petal color, these plants can be divided into eight groups, including the white, yellow, light pink, pink, red, purple, dark red, and orange” has been revised to “and these cultivars can be divided into nine flower color categories including red, pink, white, blue, purple, green, yellow, black and double color”. Thanks again.

11. Reviewer #3: In line 43,“with” should be revised to “among”.

Answer: Thank you for your comments very much. “with” has been revised to “among”. Thanks again.

12. 12. Reviewer #3:In line 51, check if all cultivars' names are in single quotation marks ('), not in double (").

Answer: We are very sorry for our carelessness. We have revised all cultivars' names to be in single quotation marks. Thank you for your comments very much.

13. 13. Reviewer #3:In line 58(HPLC), you need to explain all abbreviations (except for the commonly understandable such as DNA) at the very first mention.

Answer: We are very sorry for our carelessness. We have explained all abbreviations at the first mention in the manuscript, please check below.

(1) in line 60, “HPLC” has been revised to “high-performance liquid chromatography (HPLC)”.

(2) in line 25, “PlANS and PlUF3GT” has been revised to “thocyanidin synthase gene (PlANS) and UDP-Glucose: flavonoid 3-O-glucosyltransferase gene (PlUF3GT)”.

14. 14. Reviewer #3:In line 63,'Violet' stands for the other plant genus (Viola), not for Matthiola, which can be called 'common stock' or 'hoary stoack'. Actually, Latin name is sufficient to understand, what plant is considered.

Answer: Thank you for your comments very much. “Violet (Matthiola incana).” has been revised to “Matthiola incana”. Thanks again.

15. 15. Reviewer #3:In line 64,Why are you discussing these taxa, which are not even closely related to Paeonia?

Answer: Thank you for your comments very much. We discussed these taxa to validate our previous description “Previous studies have already identified anthocyanins in various plants, and often in some species with red flowers.”. Thanks again.

16. 16. Reviewer #3:In line 64, I cannot understand what it means regarding a chemical substance.

Answer: Thank you for your comments very much. This sentence means the derivatives of delphinidin and cyaniding were more complex in the red Nymphaea than those in blue ones. And “complicated” has been revised to “complex”. Thanks again.

17. 17. Reviewer #3:In line 75,“e” should be revised to “the”.

Answer: We are very sorry for our carelessness. “e” has been revised to “the”. Thank you for your comments very much.

18. 18. Reviewer #3:In line 76(catalyzed by), anthocyanins themselves cannot be catalyzed, only reactions.

Answer: We are very sorry for our carelessness. “catalyzed” has been revised to “reacted”. Thank you for your comments very much.

19. 19. Reviewer #3:In line 81 (Camellia sinensis), You should either provide authors' names with every Latin name or avoid them everywhere.

Answer: We are very sorry for our carelessness. We have checked all Latin names and deleted authors' names. Thank you for your comments very much.

20. 20. Reviewer #3:In line 83,here and in similar contexts, 'flower' is hardly a good decision, as it means a plant organ. If you want to write about plants with red flowers, you should better use 'red-flowered forms' etc.

Answer: We are very sorry for our carelessness. “red flowers” has been revised to “red-flowered forms”. Thank you for your comments very much.

21. 21. Reviewer #3:In line 89(synthesize), genes do not synthesize pigments. Genes encode enzymes, which catalyze a biosynthesis of pigments.

Answer: We are very sorry for our carelessness. “Our previous studies have identified that high expressions of PlDFR, PlANS and PlUF3GT synthesize colored pigments in a purplish-red P. lactiflora cv. ‘Hongyan Zhenghui’ [26].” Has been revised to “Previously, we identified that PlDFR, PlANS and PlUF3GT were highly expressed in a purplish-red P. lactiflora cv. ‘Hongyan Zhenghui’ [26].”. Thank you for your comments very much.

22. 22. Reviewer #3:In line 89, 'Colored pigments' is an excessive construction. Pigments are never colorless by their definition.

Answer: We are very sorry for our carelessness. We have deleted it. Thank you for your comments very much.

23. 23. Reviewer #3:In line 92,all these descriptions should be moved to the 'Materials and Methods' section.

Answer: Thank you for your comments very much. These descriptions are intended to illustrate the purpose and specific contents of this study, as well as the significance of the results. Thanks again.

24. 24. Reviewer #3:In line 94(were analyzed), not anthocyanins but their quantities.

Answer: We are very sorry for our carelessness. “analyzed” has been revised to “quantified”. Thank you for your comments very much.

25. 25. Reviewer #3:In line 98(been isolated), the expression levels cannot be 'isolated'.

Answer: We are very sorry for our carelessness. “isolated” has been revised to “detected”. Thank you for your comments very much.

26. 26. Reviewer #3:In Figure 1,I recommend you to give cultivar's name only once (e.g., at the left image in each row), not in every photo.

Answer: Answer: Thank you for your comments very much. We have revised the Figure 1, please check it. Thanks again.

 

27. 27. Reviewer #3:In line 112,quote Fig. 1 here.

Answer: Answer: We are very sorry for our carelessness. We have quoted Figure 1 quote Fig. 1. Thank you for your comments very much.

28. 28. Reviewer #3:In line 122,if possible, please cite a work where this method is described or describe it in more details.

Answer: We are very sorry for our carelessness. Because this method is often used in China, we did not add it. And the reference “Zhao, D.Q., Sheng, Y.L., Zhou, C.H. Method of non-destructive measurement research on leaf area and fruit fresh weight of Ginkgo biloba. North. Hortic. 2010, 7, 28-30. (doi: 10.11937/bfyy.201007009)” has been added. Thank you for your comments very much.

29. 29. Reviewer #3:In line 205, Previously (lines 191-192) you stated that you used S.D., not S.E. What is given in this table? If S.E., they are not correct indicator of variation (for explanation, see: Lang (2004) Twenty statistical errors even YOU can find in biomedical research articles. Croat. Med. J. 45(4): 361-370).

Answer: We are very sorry for our carelessness. It should be S.D., and “SE” has been revised to “SDs”. Thank you for your comments very much.

30. 30. Reviewer #3:In line 207,“higher” should be revised to “bigger”.

Answer: Thank you for your comments very much. “higher” has been revised to “bigger”. Thanks again.

31. 31. Reviewer #3:In Figure 2,it is better to use only one title ('Developmental stage') for horisontal axes in both graphs.

Answer: Thank you for your comments very much. According to your comments, we have revised the Figure 2, please check it. Thanks again.

32. 32. Reviewer #3:In line 214, I don't think you need to explain these abbreviations in every figure's caption. The best is to write something like 'for stage designations, see Fig. 2' in all subsequent cases.

Answer: Thank you for your comments very much. According to your comments, “For stage designations, see Figure 2.” has been added in Figures 3-6. Thanks again.

33. 33. Reviewer #3:In line 216, “SE” should be revised to “SDs”.

Answer: We are very sorry for our carelessness. “SE” has been revised to “SDs”. Thank you for your comments very much.

34. 34. Reviewer #3:In Figure 3, left vertical axis: use degree sign, not a uppercase zero.Horisontal axis: use a single title ('Developmental stage').

Answer: Thank you for your comments very much. According to your comments, we have revised the Figure 3, please check it. Thanks again.

35. 35. Reviewer #3:In line 234,“Analysis of the anthocyanins and the flavones as well as flavonols” should be revised to “Analysis of the anthocyanins, flavones, and flavonols”.

Answer: We are very sorry for our carelessness. “Analysis of the anthocyanins and the flavones as well as flavonols” has been revised to “Analysis of the anthocyanins, flavones, and flavonols”. Thank you for your comments very much.

36. 36. Reviewer #3:In lines 235-236, check through the whole text: such details should remain only in the 'Materials and Methods' section. There is no need to repeat them in 'Results'.

Answer: Thank you for your comments very much. The sentence “Anthocyanins, as well as flavones and flavonols extracted from the petals, were analyzed by HPLC–PDA–MSⁿ, as shown in Figure 4.” has been deleted. Thanks again.

37. 37. Reviewer #3:In line 243,“molecular ion”?

Answer: Thank you for your comments very much. “molecular ion” is right, which is used in previous study (Jia et al., Scientia Horticulturae, 2008, 117: 167-173). Please check below. Thanks again.

 

38. 38. Reviewer #3:In Figure 4, do you need such long horisontal axis? Why not to shorten it till, say, 15 or 20 min?

Answer: Thank you for your comments very much. Because in our previous study, an anthocyanin component was detected at 33 min (please check below figure) (Wu et al., Journal of Integrative Agriculture 2016, 15: 2023-2031), we extended the time in this study. Thanks again.

 

39. 39. Reviewer #3:In line 261, “should” should be deleted.

Answer: We are very sorry for our carelessness. “should” has been deleted. Thank you for your comments very much.

40. 40. Reviewer #3:In line 278, “gene”should be revised to “genes”.

Answer: We are very sorry for our carelessness. “gene” has been revised to “genes”. Thank you for your comments very much.

41. 41. Reviewer #3:In Figure 6,indicate statistically reliable differences.

Answer: We are very sorry for our carelessness. And the statistical analysis has been added in Figure 6, please check below. Thank you for your comments very much.

 

42. 42. Reviewer #3:In line 310 (Sugar is the product of photosynthesis), in each plant organ, sugars can be synthesized in the course of photosynthesis, but may be also transferred from other organs (e.g. those accumulating starch etc.). Petals themselves are (almost) not photosynthesizing, so their carbohydrate content is most probably dependent on other organs.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”. Thanks again.

43. 43. Reviewer #3:In line 314, this percentage is not indicative. The principal point is whether these differences are statistically significant or not.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”. Thanks again.

44. 44. Reviewer #3:In line 317,“a lunch break”?

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”. Thanks again.

45. 45. Reviewer #3:In line 336, (In Violets(Matthiola incana)), not violets, see in the beginning. There is no need to redescribe all these findings, as you have already listed all them in the Introduction.

Answer: Thank you for your comments very much. We have deleted “In Violets (Matthiola incana), the major anthocyanins in red-purple, red, and peach flowers were Cy3G5G, Pg3G5G, and Pg3G, separately [16].”. Thanks again.

46. 46. Reviewer #3:In lines 361-363, “Our lab has isolated nine anthocyanin biosynthetic genes in P. lactiflora, including PlPAL, PlCHS, PlCHI, PlF3H, PlF3'H, PlANS, PlDFR, PlUF3GT and PlUF5GT [26].”Again, you have already told about this before.

Answer: Thank you for your comments very much. We have deleted “Our lab has isolated nine anthocyanin biosynthetic genes in P. lactiflora, including PlPAL, PlCHS, PlCHI, PlF3H, PlF3'H, PlANS, PlDFR, PlUF3GT and PlUF5GT [26].” Thanks again.

47. 47. Reviewer #3:In line 374,“distinct” should be deleted.

Answer: We are very sorry for our carelessness. “distinct” has been deleted. Thank you for your comments very much.

48. 48. Reviewer #3:In line 377,“regulate” should be revised to “encode”.

Answer: We are very sorry for our carelessness. “regulate” has been revised to “encode”. Thank you for your comments very much.

49. 49. Reviewer #3:In lines 376-377, does 'a series of enzymes' lead to 'the accumulation' of pigments? I cannot resolve what exactly you have shown with your expression studies.

Answer: We are very sorry for our carelessness. “These results suggested that both the early anthocyanin biosynthetic pathway genes and the late ones participate in the pathway and encode a series of enzymes which leads to the accumulation of anthocyanins.” has been revised to “These results suggested that the expression of the structural genes in anthocyanin biosynthetic pathway is directly associated with the accumulation of anthocyanins.”. Thank you for your comments very much.

50. Reviewer #3: In line 381, you need to elaborate this part carefully. At the moment, there is no clarity how expression levels of certain genes correspond to contents of different substances.

Answer: We are very sorry for our carelessness. “Subsequently, a large number of intermediate products, such as flavanones, dihydroflavonols, flavones, and flavonols were produced although with the lower expression levels of PlC4H, PlCHS, PlCHI, PlF3H.” has been revised to “Subsequently, a large number of intermediate products were produced although with the lower expression levels of PlC4H, PlCHS, PlCHI, PlF3H.”. Thank you for your comments very much.

51. 51. Reviewer #3:In lines 386-390, rephrase this sentence, it is very difficult to understand.

Answer: Thank you for your comments very much. The sentence “The situation was On the contrary in ‘Qingwen’ the low expression levels of early genes PlPAL and PlF3'H caused: the upstream substances to be insufficient and due to the low expression levels of the late genes PlANS and PlUF3GT, very few upstream substances were transformed into anthocyanins, imparting a light pink color to the petals.” has been revised to “Whereas in ‘Qingwen’, the upstream substances was insufficient because of the low expression levels of early genes PlPAL and PlF3'H. And the formation of anthocyanins was inhibited because of the low expression levels of the late genes PlANS and PlUF3GT, which resulted in a light pink color of petals.”. Thanks again.

52. 52. Reviewer #3:In line 391,were they identified in this quality by you? Or are these genes already known as key regulators of anthocyanin biosynthesis?

Answer: Thank you for your comments very much. PlPAL, PlF3'H, PlANS, and PlUF3GT were identified as critical genes based on the expression patterns between the two cultivars. And “In conclusion,” has been revised to “It could be seen that”. Thanks again.

53. 53. Reviewer #3:In line 403, this statement is a huge over-generalization.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

54. 54. Reviewer #3:In line 409,genes do not catalyze anything.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

55. 55. Reviewer #3:In line 415, italicize Latin name (but not the multiplication sign).

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

56. 56. Reviewer #3:In line 415, you never introduced the whole generic name before.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

57. 57. Reviewer #3:In line 420, “indexes”should be revised to “indices”.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

58. 58. Reviewer #3:All this story of correlation between sugars' and anthocyanins' contents is very shaky. As you of course remember, correllation does not (always) mean causation, so all these statistical findings may have no biological sense. To prove it, you need to conduct additional studies. I suggest you either to completely omit all this sugar-related part from your paper or to describe it with a maximum accuracy. At the moment, you don't seem to have enough basis for founded statements.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

59. 59. Reviewer #3:Or was not.These cultivars you studied are completely different, they are not isogenous lines. They may have different anthocyanin content and different efficiency of photosynthesis without any connection between these two features.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

60. 60. Reviewer #3:In line 437 (floral nectar), have you studied this feature?I doubt that these two cultivars do secrete nectar in any detectable quantities, as they both are 'double-flowering', with flowers composed of supernumerary petals. Forms like this are usually nectarless. Anyway, you need to check this - but you cannot use the untested feature as a proof.I guess, I all studies on nectar secretion in peonies are dedicated to wild-type flowers, not to 'double-flowering' cultivars.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

61. 61. Reviewer #3:In lines 446-447, Previously you stated that the anthocyanin composition was identical in two studied cultivars.I recommend you to rephrase Conclusions to narrow them down to your own studies. Actually, you cannot exclude that there may be two peony cultivars having different red colors because of different anthocyanin composition, not only content.

Answer: Thank you for your comments very much. According to your comments, the conclusion has been revised to “In P. lactiflora cultivars ‘Dafugui’ and ‘Qingwen’, the same compositions of the an-thocyanins were found, and the depth of the red color was determined by the anthocya-nin content. Low expressions of the early genes (especially PlPAL and PlF3'H) resulted in insufficient upstream substances, and the formation of anthocyanins was inhibited because of the low expression levels of the late genes (especially PlANS and PlUF3GT), which resulted in a light pink color of petals. This study can serve as a theoretical basis for breeding more red flowers using genetic engineering techniques to cater to the preferences of consumers.”. Thanks again.

62. 62. Reviewer #3:In lines 448-449,it is an over-generalization again. This statement is trivial.

Answer: Thank you for your comments very much. According to your comments, the conclusion has been revised to “In P. lactiflora cultivars ‘Dafugui’ and ‘Qingwen’, the same compositions of the an-thocyanins were found, and the depth of the red color was determined by the anthocya-nin content. Low expressions of the early genes (especially PlPAL and PlF3'H) resulted in insufficient upstream substances, and the formation of anthocyanins was inhibited because of the low expression levels of the late genes (especially PlANS and PlUF3GT), which resulted in a light pink color of petals. This study can serve as a theoretical basis for breeding more red flowers using genetic engineering techniques to cater to the preferences of consumers.”. Thanks again.

63. 63. Reviewer #3:In line 454,“the formation of anthocyanin involves the consumption of sugars”, you have not proved it.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

64. 64. Reviewer #3:In line 455,“sugar content can affect the anthocyanin accumulation in flowers”, you need to prove it.

Answer: Thank you for your comments very much. According to your comments, we have deleted “3.5. Soluble sugar content and photosynthetic characteristics”, and the corresponding discussion also has been deleted. Thanks again.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

1. Authors’ proofreading made the ambiguous interpretation of results. In this study, the authors indicated the flavonoid biosynthesis pathway in Peony crops in a previous study (Supplementary Figure 1). However, the authors detected the flavonols and flavanones, which were not identified in previous studies, were detected in peony flowers (Figure 4 and Table 1). I asked about the expression analysis of the FLS/FNS gene, which is crucial for the synthesis of flavonol and flavanones in plants. But authors mentioned that the FLS/FNS did not exist in peony. Do the authors think about how to accumulate the flavonols and flavanones in peony flowers without these genes expression? This manuscript should be improved via scientific interpretation of the research results and more experiments for analyzing gene expression data and then given the scientific meaning to readers.
2. Authors should write the lined in table 1
3. In the figure 5, please put the legend (related in cultivar) inside the figure, and delete the cultivar legend box.
4. Based on the journal policy, the edited manuscript should be written by tracking in red font. But it was not followed the policy, and just shown in the highlighted. Please rewrite the manuscript to confirm the corrections.

Author Response

Here below is our description on revision according to reviewers’ comments.

Reviewer’s comments and suggestions 2:

1. Reviewer #2:English language and style:Moderate English changes required.

Answer: Thank you for your comments very much. The language of this manuscript has been edited by “MDPI's Author Services”, and we carefully proof-read the manuscript to minimize typographical and grammatical errors, please check below. Thanks again.

 

 

 

2. 2. Reviewer #2:Authors’ proofreading made the ambiguous interpretation of results. In this study, the authors indicated the flavonoid biosynthesis pathway in Peony crops in a previous study (Supplementary Figure 1). However, the authors detected the flavonols and flavanones, which were not identified in previous studies, were detected in peony flowers (Figure 4 and Table 1). I asked about the expression analysis of the FLS/FNS gene, which is crucial for the synthesis of flavonol and flavanones in plants. But authors mentioned that the FLS/FNS did not exist in peony. Do the authors think about how to accumulate the flavonols and flavanones in peony flowers without these genes expression? This manuscript should be improved via scientific interpretation of the research results and more experiments for analyzing gene expression data and then given the scientific meaning to readers.

Answer: Thank you for your comments very much, and we are very sorry for our vague description. In those two papers (Molecular Biology Reports, 2012, 39: 11263-11275; Electronic Journal of Biotechnology, 2012, 15: 6), there is no information about the role of FLS/FNS in the biosynthetic pathway. And according to your comments, we searched the NCBI database and found one FLS gene (GenBank accession number KM259902), but no FNS gene was found. And we examined the expression level of FLS gene in these two cultivars, and its expression level was higher in the pink cultivar than in the red cultivar, and it have been added in the manuscript. Please check below. And your previous comments will be of great help to our future research. Thanks again.

3. 3. Reviewer #2:Authors should write the lined in table 1.

Answer: We are very sorry for our carelessness. We have revised the table 1 according to your comments, please check below. Thank you for your comments very much.

Peak	tR (min)	λmax(nm)	MS1 (m/z)	MS2 (m/z)	Identification
a1	3.01	235, 275, 515	611.2	448.9, 287.2	Cyanidin-3,5-di-O-glucoside [17,29]
a2	4.80	275, 510	625.2	462.9, 301.1	Peonidin-3,5-di-O-glucoside [17]
f1	7.31	235, 315	627.0	464.9, 303.3	Quercetin di-hexoside [31]
f2	10.32	265, 345	611.1	449.1, 287.3	Kaempferol di-hexoside [32]
f3	11.57	255, 355	641.0	478.9, 317.0	Isorhamnetin di-hexoside [32]
f4	21.75	235, 370	465.1	303.2	Quercetin-3-O-rhamnoside [33]
f5	29.19	230, 265, 365	449.1	287.2	Luteolin-7-O-glucoside [33]

4. 4. Reviewer #2:In the figure 5, please put the legend (related in cultivar) inside the figure, and delete the cultivar legend box.

Answer: Thank you for your comments very much. We have revised the figure 5 according to your comments, please check below. Thanks again.

 

5. 5. Reviewer #2:Based on the journal policy, the edited manuscript should be written by tracking in red font. But it was not followed the policy, and just shown in the highlighted. Please rewrite the manuscript to confirm the corrections.

Answer: We are very sorry for our carelessness. We rewrote the manuscript by tracking in red font, please check the manuscript. Thank you for your comments very much.

 

Reviewer’s comments and suggestions 3:

1. Reviewer #3: After considering all (or most of) suggestions, this paper can be recommended for publication.

Answer: Thank you very much for your hard work on this manuscript. Your previous comments will be of great help to our future research. Thanks again.

2. 2. Reviewer #3: English language and style:English language and style are fine/minor spell check required.

Answer: Thank you for your comments very much. The language of this manuscript has been edited by “MDPI's Author Services”, and we carefully proof-read the manuscript to minimize typographical and grammatical errors, please check below. Thanks again.

 

(1) In line 2, “high temperature stress” has been revised to “high-temperature stress”.

 

 

 

 

 

 

Thank you and all the reviewers for the kind advice. Should you have any questions, please contact us without hesitate. I prefer communication by email and I'm looking forward to hearing from you soon.

 

Sincerely yours,

Jun Tao

College of Horticulture and Plant Protection, Yangzhou University,

Yangzhou 225009, PR China

Email: [email protected]

Tel: +86-514-87997219

Fax: +86-514-87347537

Author Response File: Author Response.doc

Reviewer 3 Report

After considering all (or most of) suggestions, this paper can be recommended for publication.

Author Response

cHere below is our description on revision according to reviewers’ comments.

Reviewer’s comments and suggestions 3:

1. Reviewer #3: After considering all (or most of) suggestions, this paper can be recommended for publication.

Answer: Thank you very much for your hard work on this manuscript. Your previous comments will be of great help to our future research. Thanks again.

2. 2. Reviewer #3: English language and style:English language and style are fine/minor spell check required.

Answer: Thank you for your comments very much. The language of this manuscript has been edited by “MDPI's Author Services”, and we carefully proof-read the manuscript to minimize typographical and grammatical errors, please check below. Thanks again.

 

 

 

Thank you and all the reviewers for the kind advice. Should you have any questions, please contact us without hesitate. I prefer communication by email and I'm looking forward to hearing from you soon.

 

Sincerely yours,

Jun Tao

College of Horticulture and Plant Protection, Yangzhou University,

Yangzhou 225009, PR China

Email: [email protected]

Tel: +86-514-87997219

Fax: +86-514-87347537

Author Response File: Author Response.doc