Identification of Late Flavonoid Biosynthesis Genes of Moso Bamboo Reveals the Potential Function of PeANR4 Involved in Osmotic and Salt Stress

Round 1

Reviewer 1 Report

 

 The paper is regarding flavonoid and functional characterization of NAR4 gene in Bamboo. It is good study and a lot of data generated. I could not find that Data is submitted in any repository or any statement regarding availability of data. I pointed out some query and suggestion. 

·         How PAs played is role in DNA protection?

·         The reason of using specifically Oryza sativa as reference specie?

·         What is the purpose of inserting promoter fragment of PeANR4 into pHIS2 vector while it was initially cloned into PGADT7-Rec2 vector?

Grammatical and sentence structure errors

Line 11, 55, 341, 344, 375, 404

Line 137: write days or mention in abbreviation when it is used first time in text e.g.  21 d (days)

Line 332: The germination rate the transgenic lines 

Grammatical and sentence structure errors

Line 11, 55, 341, 344, 375, 404

Line 137: write days or mention in abbreviation when it is used first time in text e.g.  21 d (days)

Line 332: The germination rate the transgenic lines 

 

Author Response

Dear Editor and Reviewers,
Thank you very much for giving us the opportunity to revise our manuscript entitled ‘Identification of Late Flavonoid Biosynthesis Genes of Moso Bamboo Reveals the Potential Function of PeANR4 Involved in Drought and Salt Stress (manuscript ID: 2442520)’. We have followed you and the reviewer’s suggestions and made revisions carefully. The revised manuscript has been resubmitted. Please check the revised manuscript in details, which is marked in red.
We hope all these corrections and revisions will be satisfactory and hope that the revised manuscript will be acceptable for the publication in forests.
We thank the reviewers for their concerns and hard work. The responses to the reviewers are listed below.
---------------------------------------------------------------------------------------------------------------------------------
Responds to the Reviewers’ comments:
Reviewer 1 Comments to Author:
The paper is regarding flavonoid and functional characterization of ANR4 gene in Bamboo. It is good study and a lot of data generated. I could not find that Data is submitted in any repository or any statement regarding availability of data. I pointed out some query and suggestion.
Answer: Thank you for your comment and suggestion. The available transcriptome data of previous studies were used for gene tissue specific and spatiotemporal expression analyses, respectively, including the data generated from 26 different tissues of moso bamboo (Zhao et al.; 2018), abiotic stresses (drought and cold) (Huang et al.; 2016) and those produced by the 13th internode of moso bamboo shoots (Yang et al.; 2021). The accession number were SRS1847048-SRS1847073, SRS1759772 and PRJNA673565 in NCBI Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra Accessed Dec. 2022) correspondingly.

Reference:
27. Zhao, H.; Gao, Z.; Wang, L.; Wang, J.; Wang, S.; Fei, B.; Chen, C.; Shi, C.; Liu, X.; Zhang, H.; et al. Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis). Gigascience 2018, 7, giy115.
28. Huang, Z.; Jin, S.H.; Guo, H.D.; Zhong, X.J.; He, J.; Li, X.; Jiang, M.Y.; Yu, X.F.; Long, H.; Ma, M.D.; et al. Genome-wide identification and characterization of TIFY family genes in moso bamboo (Phyllostachys edulis) and expression profiling analysis under dehydration and cold stresses. Peer J. 2016, 4, e2620.
29. Yang, K.; Li, L.; Lou, Y.; Zhu, C.; Li, X.; Gao, Z. A regulatory network driving shoot lignification in rapidly growing bamboo. Plant Physiol. 2021, 187, 900-916.

1. How PAs played is role in DNA protection?
Answer: Thank you for your comments. PAs play roles in DNA protection by reducing oxidative stress (Bagchi et al., 2000), modulating the DNA repair gene expression (Bakheet et al., 2016), and activating the Nrf2/ARE cell signaling (Suraweera et al., 2023), etc. 

Reference: 
Bagchi D.; Bagchi M.; Stohs S.J.; Das D.K.; Ray S.D, Kuszynski C.A.; Joshi S.S.; Pruess H.G. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology. 2000, 148(2-3): 187-197.
Bakheet, S.A.; Alhuraishi, A.M.; Al-Harbi, N.O.; Al-Hosaini, K.A.; Al-Sharary, S.D.; Attia, M.M.; Alhoshani, A.R.; Al-Shabanah, O.A.; Al-Harbi, M.M.; Imam, F.; et al. Alleviation of aflatoxin B1-induced genomic damage by proanthocyanidins via modulation of DNA repair. J. Biochem. Mol. Toxicol. 2016, 30, 559-566.
Suraweera T.L.; Merlin J.P.J.; Dellaire G.; Xu Z.; Rupasinghe H.P.V. Genistein and Procyanidin B2 Reduce Carcinogen-Induced Reactive Oxygen Species and DNA Damage through the Activation of Nrf2/ARE Cell Signaling in Bronchial Epithelial Cells In Vitro. Int. J. Mol. Sci. 2023, 24(4): 3676.

2. The reason of using specifically Oryza sativa as reference species?
Answer: Thank you for your comments. As both Oryza sativa and moso bamboo are members of the grass family Poaceae, they have a close genetic relationship (Peng et al., 2-13). Additionally, O. sativa is a model plant with high quality genome and has been extensively well-studied in various aspects.

Reference: 
Peng Z.; Lu Y.; Li L.; Zhao Q.; Feng Q.; Gao Z.; Lu H.; Hu T.; Yao N.; Liu K.Y.; et al. The draft genome of the fast-growing non-timber forest species moso bamboo (Phyllostachys heterocycla). Nat. Genet. 2013, 45: 456–461.e4612.

3. What is the purpose of inserting promoter fragment of PeANR4 into pHIS2 vector while it was initially cloned into pGADT7-Rec2 vector?
Answer: Thank you for your comments. Our purpose is to verify whether the transcription factor can bind to the promoter of PeANR4 using yeast one-hybrid assays. Therefore, TF was linked into pGADT7-Rec2 vector, and the promoter of PeANR4 was inserted into the pHIS2 vector. 

4. Grammatical and sentence structure errors
Line 11, 55, 341, 344, 375, 404
Answer: Thank you for the suggestion. We have revised them as follows. 
Line 11: The sentence is revised to “Flavonoids are important secondary metabolites in plants, and their biosynthesis includes various enzymes.”
Line 55: The sentence is revised to “Research has shown that flavonoids in bamboo leaves possess strong antioxidant capabilities, which can be used for immune modulation, anti-inflammation, antidepressant, and lipid-lowering [18-20].” 
Line 341: The sentence is revised to “but overall, roots of OE-1 and OE-2 plants suffered less inhibition than WT and ban, those …”
Line 344: The sentence is revised to “… those of ban were the shortest under treatments of mannitol….”
Line 375: The sentence is revised to “We also found that PeDFR1, only one copy identified in bamboo, clustered in the same clade with OsDFR, indicating PeDFR1 had similar function to OsDFR.”
Line 404: The sentence is revised to “…the promoter of PeANR4, but further studies are needed to confirm this transcriptional regulation in bamboo.”

Line 137: write days or mention in abbreviation when it is used first time in text e.g. 21 d (days)
Answer: Thank you for the suggestion. We have revised them. Please see the details in the revised manuscript.

Line 332: The germination rate the transgenic lines 
Answer: Thank you for the suggestion. We have revised it as follows.
“The germination rates of the transgenic lines”.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

Reviewer comments forests-2442520

The manuscript entitled „Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in drought and salt stress“ represents a valuble study aimed at an investigation of late flavonoid biosynthesis genes (LFBGs), namely PeANR4, in bamboo subjected to salt and osmotic stress. Functional analysis of PeANR4 was performed by its overexpression in A. thaliana. The manuscript contains novel and very important data on 24 late flavonoid biosynthesis genes (LFBGs) in moso bamboo which are worth publishing.

However, I have several major comments on the present version of the manuscript which has to be addressed by the authors prior to the manuscript publication.

1/ Abbreviations list. There are several abbreviations related to flavonoid biosynthesis mentioned in the text out of which only some of them are adequately explained. Moreover, some of the used abbreviations such as PAs for condensed tannins (also known as polyflavonoid tannins) are confusing since „PAs“ is commonly used for polyamines which are entirely different biomolecules. I therefore recommend the authors to use other abbreviation than „PAs“ for polyflavonoid tannins. I recommend the authors to add a separate Abbreviations list with appropriate explanation in the manuscript.

2/ Materials and methods, plant material source: The source of moso bamboo seedlings (line 95) has to be specified, i.e., it has to be given from which institution or natural habitat they were obtained.

3/ Materials and methods: The date of access to the whole genome database of bamboo in the BamGDB database has to be added.

4/ Materials and methods, Statistical analysis: A kind of a post-hoc test of multiple comparisons following ANOVA analysis has to be given, i.e., which kind of a post-hoc test was used for determination of significant differences has to be specified.

5/ Results:

Figure 1. In Figure 1 providing a phylogenetic tree of phylogenetic analysis of LAR, ANS, ANR, IFR and DFR proteins, appropriate statistical evaluation has to be added, i.e., bootstrap values at nodes indicating probability of the branching per 1000 replicates o ran appropriate scale bar. In the present form, the phylogenetic tree is just an illustrative scheme without any scientific value.

6/ In Figure 3, 6, 7, and 8, the kind of a post-hoc test used for the determination of significant differences has to be given in the figure legends.

7/ Terminology:

Mannitol treatment has to be termed as „osmotic stress“ , not „drought stress“ which means a water deficit in soil. Please, correct this term in the whole manuscript.

Line 365: Correct the chemical name „chalcone synthesis“ (not „chalone synthesis“).

8/ Formal comments on the text:

Materials and methods, line 87: Add a comma between the words „analyses“ and „respectively.“

Line 116: An appropriate reference has to be added to the following statement: „Yeast one-hybrid (Y1H) assays were performed using yeast strain Y187 according to the manufactiurer´s instructions.“

Line 127: Modify the heading as follows: „2.6. Arabidopsis thaliana transformation“.

Line 254: Modify the statement as follows: „…that four genes  revealed the increased expression of all four genes.“

Line 268: Correct the word form in „two numbers“ (not „two number“).

Line 269: Add a comma between the words „peDFR1“ and „respectively“.

Line 275: Modify the statement as follows: „…which indicated that it may be regulated by multiple types of TFs and Pas synthesis to form a complex regulátory network.“

Line 291: Add a space between „TFs“ and „could“.

Line 332: Add the word „of“ in the statement „The germination rates of the transgenic lines…“

Line 340: Modify the word form „obviouslyů to „obvious“ in the statement „….the inhibition of of root growth became more obvious…“

In Figure 6 legend, line 320-321, please remove „OE-3 and OE-4“ since only data from OE-1 and oE-2 lines are presented in the figure.

In Figure 7 legend, please, correct the description as follows: „Germination phenotype (a,b), and germination rate of Arabidopsis under osmotic and salt stress. Asterisks indicate the significant level   (…..) as determined by …. Test. Scale bar: 1 cm?? „ Information on the scale bar has to be added.

Conclusion, line 438: Modify the statement as follows: „…DEGs asociated with different shoot heights…“

Line 442: replace the word „of“ with „in“ in the statement „…PA synthesis in transgenic Arabidopsis plants…“

Line 445: Modify the statement as follows: „…and has important practical signifikance for the development of flavonoid-rich bamboo.“

 

Final recommendation: Reconsider after a major revision.

 

 

 

 

Dear Authors,

 

Reviewer comments forests-2442520

The manuscript entitled „Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in drought and salt stress“ represents a valuble study aimed at an investigation of late flavonoid biosynthesis genes (LFBGs), namely PeANR4, in bamboo subjected to salt and osmotic stress. Functional analysis of PeANR4 was performed by its overexpression in A. thaliana. The manuscript contains novel and very important data on 24 late flavonoid biosynthesis genes (LFBGs) in moso bamboo which are worth publishing.

However, I have several major comments on the present version of the manuscript which has to be addressed by the authors prior to the manuscript publication.

1/ Abbreviations list. There are several abbreviations related to flavonoid biosynthesis mentioned in the text out of which only some of them are adequately explained. Moreover, some of the used abbreviations such as PAs for condensed tannins (also known as polyflavonoid tannins) are confusing since „PAs“ is commonly used for polyamines which are entirely different biomolecules. I therefore recommend the authors to use other abbreviation than „PAs“ for polyflavonoid tannins. I recommend the authors to add a separate Abbreviations list with appropriate explanation in the manuscript.

2/ Materials and methods, plant material source: The source of moso bamboo seedlings (line 95) has to be specified, i.e., it has to be given from which institution or natural habitat they were obtained.

3/ Materials and methods: The date of access to the whole genome database of bamboo in the BamGDB database has to be added.

4/ Materials and methods, Statistical analysis: A kind of a post-hoc test of multiple comparisons following ANOVA analysis has to be given, i.e., which kind of a post-hoc test was used for determination of significant differences has to be specified.

5/ Results:

Figure 1. In Figure 1 providing a phylogenetic tree of phylogenetic analysis of LAR, ANS, ANR, IFR and DFR proteins, appropriate statistical evaluation has to be added, i.e., bootstrap values at nodes indicating probability of the branching per 1000 replicates o ran appropriate scale bar. In the present form, the phylogenetic tree is just an illustrative scheme without any scientific value.

6/ In Figure 3, 6, 7, and 8, the kind of a post-hoc test used for the determination of significant differences has to be given in the figure legends.

7/ Terminology:

Mannitol treatment has to be termed as „osmotic stress“ , not „drought stress“ which means a water deficit in soil. Please, correct this term in the whole manuscript.

Line 365: Correct the chemical name „chalcone synthesis“ (not „chalone synthesis“).

8/ Formal comments on the text:

Materials and methods, line 87: Add a comma between the words „analyses“ and „respectively.“

Line 116: An appropriate reference has to be added to the following statement: „Yeast one-hybrid (Y1H) assays were performed using yeast strain Y187 according to the manufactiurer´s instructions.“

Line 127: Modify the heading as follows: „2.6. Arabidopsis thaliana transformation“.

Line 254: Modify the statement as follows: „…that four genes  revealed the increased expression of all four genes.“

Line 268: Correct the word form in „two numbers“ (not „two number“).

Line 269: Add a comma between the words „peDFR1“ and „respectively“.

Line 275: Modify the statement as follows: „…which indicated that it may be regulated by multiple types of TFs and Pas synthesis to form a complex regulátory network.“

Line 291: Add a space between „TFs“ and „could“.

Line 332: Add the word „of“ in the statement „The germination rates of the transgenic lines…“

Line 340: Modify the word form „obviouslyů to „obvious“ in the statement „….the inhibition of of root growth became more obvious…“

In Figure 6 legend, line 320-321, please remove „OE-3 and OE-4“ since only data from OE-1 and oE-2 lines are presented in the figure.

In Figure 7 legend, please, correct the description as follows: „Germination phenotype (a,b), and germination rate of Arabidopsis under osmotic and salt stress. Asterisks indicate the significant level   (…..) as determined by …. Test. Scale bar: 1 cm?? „ Information on the scale bar has to be added.

Conclusion, line 438: Modify the statement as follows: „…DEGs asociated with different shoot heights…“

Line 442: replace the word „of“ with „in“ in the statement „…PA synthesis in transgenic Arabidopsis plants…“

Line 445: Modify the statement as follows: „…and has important practical signifikance for the development of flavonoid-rich bamboo.“

 

Final recommendation: Reconsider after a major revision.

 

 

Author Response

Dear Editor and Reviewers,
Thank you very much for giving us the opportunity to revise our manuscript entitled ‘Identification of Late Flavonoid Biosynthesis Genes of Moso Bamboo Reveals the Potential Function of PeANR4 Involved in Drought and Salt Stress (manuscript ID: 2442520)’. We have followed you and the reviewer’s suggestions and made revisions carefully. The revised manuscript has been resubmitted. Please check the revised manuscript in details, which is marked in red.
We hope all these corrections and revisions will be satisfactory and hope that the revised manuscript will be acceptable for the publication in forests.
We thank the reviewers for their concerns and hard work. The responses to the reviewers are listed below.
---------------------------------------------------------------------------------------------------------------------------------
Responds to the Reviewers’ comments:
Reviewer 2 Comments to Author:
The manuscript entitled “Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in drought and salt stress” represents a valuble study aimed at an investigation of late flavonoid biosynthesis genes (LFBGs), namely PeANR4, in bamboo subjected to salt and osmotic stress. Functional analysis of PeANR4 was performed by its overexpression in A. thaliana. The manuscript contains novel and very important data on 24 late flavonoid biosynthesis genes (LFBGs) in moso bamboo which are worth publishing.
However, I have several major comments on the present version of the manuscript which has to be addressed by the authors prior to the manuscript publication.

1. Abbreviations list. There are several abbreviations related to flavonoid biosynthesis mentioned in the text out of which only some of them are adequately explained. Moreover, some of the used abbreviations such as PAs for condensed tannins (also known as polyflavonoid tannins) are confusing since “PAs” is commonly used for polyamines which are entirely different biomolecules. I therefore recommend the authors to use other abbreviation than “PAs” for polyflavonoid tannins. I recommend the authors to add a separate Abbreviations list with appropriate explanation in the manuscript.
Answer: Thank you for your comments and suggestions. We have referred some literature, in which proanthocyanidins or condensed tannins are abbreviated as PAs (Hong et al., 2017; Tan et al., 2018; Yu et al., 2022). Moreover, we have added the list to the Abbreviation section. Please see the details in the revised manuscript.

Reference:
Hong, M.; Hu, K.; Tian, T.; Li, X.; Chen, L.; Zhang, Y.; Yi, B.; Wen, J.; Ma, C.; Shen, J.; et al. Transcriptomic analysis of seed coats in yellow-seeded brassica napus reveals novel genes that influence proanthocyanidin biosynthesis. Front. plant sci. 2017, 8, 1674.
Tan L, Wang M, Kang Y, Azeem F, Zhou Z, Tuo D, María Preciado Rojo L, Khan IA, Pan Z. Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L. Molecules. 2018, 23(11): 2876.
Yu K.; Dixon R.A.; Duan C. A role for ascorbate conjugates of (+)-catechin in proanthocyanidin polymerization. Nat. Commun. 2022, 13(1): 3425.

2. Materials and methods, plant material source: The source of moso bamboo seedlings (line 95) has to be specified, i.e., it has to be given from which institution or natural habitat they were obtained.
Answer: Thank you for your comments and suggestions. We have supplemented the information in the materials and methods part as follows. 
“Moso bamboo seeds gathered from the Guangxi Province of China were sown in our lab and cultivated in a greenhouse (25 ± 2 ℃; 16 h / 8 h light / dark cycle).”

3. Materials and methods: The date of access to the whole genome database of bamboo in the BamGDB database has to be added.
Answer: Thank you for your suggestions. We have supplemented it in the materials and methods part.

Reference:
Zhao, H.; Gao, Z.; Wang, L.; Wang, J.; Wang, S.; Fei, B.; Chen, C.; Shi, C.; Liu, X.; Zhang, H.; et al. Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis). Gigascience 2018, 7, giy115.

4. Materials and methods, Statistical analysis: A kind of a post-hoc test of multiple comparisons following ANOVA analysis has to be given, i.e., which kind of a post-hoc test was used for determination of significant differences has to be specified.
Answer: Thank you for your comments. 
We have supplemented Fisher’s Least-Significant-Difference test in the statistical analysis part as follows.
 “Significant differences were determined by Fisher’s Least-Significant-Difference test and indicated at *0.01≤ p ≤ 0.05, ** p < 0.01 and ns: p > 0.05.”

5. Results:
Figure 1. In Figure 1 providing a phylogenetic tree of phylogenetic analysis of LAR, ANS, ANR, IFR and DFR proteins, appropriate statistical evaluation has to be added, i.e., bootstrap values at nodes indicating probability of the branching per 1000 replicates o ran appropriate scale bar. In the present form, the phylogenetic tree is just an illustrative scheme without any scientific value.
Answer: Thank you for the comment and suggestion. We have supplemented the bootstrap values at nodes indicating probability of the branching per 1000 replicates o ran appropriate scale bar. Please see the details in the revised manuscript.

6. In Figure 3, 6, 7, and 8, the kind of a post-hoc test used for the determination of significant differences has to be given in the figure legends.
Answer: Thank you for the suggestion. We have supplemented Fisher’s Least-Significant-Difference test in results part. Please see the details in the revised manuscript.

7. Terminology:
Mannitol treatment has to be termed as “osmotic stress” not “drought stress” which means a water deficit in soil. Please, correct this term in the whole manuscript.
Answer: Thank you for the suggestion. We have checked through the manuscript carefully and made revision to improve the text.

Line 365: Correct the chemical name “chalcone synthesis” (not “chalone synthesis”).
Answer: Thank you for the suggestion. We have revised it.

8. Formal comments on the text:
Materials and methods, line 87: Add a comma between the words “analyses” and “respectively.”
Answer: Thank you for the suggestion. We have supplemented it.

Line 116: An appropriate reference has to be added to the following statement: “Yeast one-hybrid (Y1H) assays were performed using yeast strain Y187 according to the manufacturer´s instructions.”
Answer: Thank you for the suggestion. We have supplemented it.

Reference:
29.Yang, K.; Li, L.; Lou, Y.; Zhu, C.; Li, X.; Gao, Z. A regulatory network driving shoot lignification in rapidly growing bamboo. Plant Physiol. 2021, 187, 900-916.

Line 127: Modify the heading as follows: “2.6. Arabidopsis thaliana transformation”.
Answer: Thank you for the suggestion. We have revised it.

Line 254: Modify the statement as follows: “…. that four genes revealed the increased expression of all four genes.”
Answer: Thank you for the suggestion. We have modified it.

Line 268: Correct the word form in “two numbers” (not “two number”).
Answer: Thank you for the suggestion. We have corrected it.

Line 269: Add a comma between the words “PeDFR1” and “respectively”.
Answer: Thank you for the suggestion. We have revised it.

Line 275: Modify the statement as follows: “…which indicated that it may be regulated by multiple types of TFs and PAs synthesis to form a complex regulatory network.”
Answer: Thank you for the suggestion. We have revised it.

Line 291: Add a space between “TFs” and “could”.
Answer: Thank you for the suggestion. We have supplemented it.

Line 332: Add the word “of” in the statement “The germination rates of the transgenic lines…”
Answer: Thank you for the suggestion. We have supplemented it.

Line 340: Modify the word form “obviously” to “obvious” in the statement “…. the inhibition of of root growth became more obvious…”.
Answer: Thank you for the suggestion. We have revised it.

In Figure 6 legend, line 320-321, please remove “OE-3 and OE-4” since only data from OE-1 and oE-2 lines are presented in the figure.
Answer: Thank you for the suggestion. We have revised it.

In Figure 7 legend, please, correct the description as follows: “Germination phenotype (a,b), and germination rate of Arabidopsis under osmotic and salt stress. Asterisks indicate the significant level   (…..) as determined by …. Test. Scale bar: 1 cm?”  Information on the scale bar has to be added.
Conclusion, line 438: Modify the statement as follows: “…DEGs asociated with different shoot heights…”
Answer: Thank you for the suggestion. We have revised it as follows. 
“Germination phenotype (a,b), and germination rate of Arabidopsis under osmotic and salt stress (c,d). Asterisks indicate the significant level (*0.01≤ p ≤ 0.05, ** p < 0.01, ns: p > 0.05) as determined by Fisher’s Least-Significant-Difference test. Scale bar: 1 cm.”

Line 442: replace the word “of” with “in” in the statement “…PA synthesis in transgenic Arabidopsis plants…”
Answer: Thank you for the suggestion. We have revised it.

Line 445: Modify the statement as follows: “…and has important practical significance for the development of flavonoid-rich bamboo.”
Answer: Thank you for the suggestion. We have revised it.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear Authors,

 

Reviewer comments forests-2442520.R1

The revised manuscript entitled „Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in osmotic and salt stress“ has been significanly improved by the authors in accordance with my previous comments. However, I still have some minor comments on the revised manuscript which are provided below.

In Materials and methods, regarding the drought and cold treatments, the authors just gave a reference on their previous study. This is correct; however, I still think that the basic information on the cold and drought treatments whose results are presented in the manuscript has to be given in Materials and methods, i.e., for cold, temperature and length of cold treatment have to be given, and for drought, some characteristics related to soil water content (SWC) and length of the treatment have to be given.

In Abbreviations list, I think that the abbreviations have to be ordered following English alphabet.

Formal comments related to English language and style:

Results, line 172: Add the word „in“ in the statement „3.1. Charcterization of LFBGs in moso bamboo“.

Results, Figure 3 legend, line 262: Correct the typing error in the word „level“ (not „leve“) in the statement „Asterisks indicate the significant level (**p≤0.01) as determiend by Fisher´s Least-Significant-Difference test.“

Discussion, line 382: Add „a“ preecding the word „similar“ in the statement „..indicating PeDFR1 had a similar function to OsDFR.“

Conclusions, line 440: I do not understand the meaning of the statement „Bamboo is an important source of flavonoids with great values…?“ – wheat does it mean „great values“? Maybe it should be modified as follows: „Bamboo is an important source of flavonoids with great economic values….“

Conclusions, line 444: Replace the verb „had“ by „are“ in the statement „There are six types of TF binding sites in the promoters of these DEGs,…“

 

Final recommendation: Accept after a minor revision.

 

Dear Authors,

 

Reviewer comments forests-2442520.R1

The revised manuscript entitled „Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in osmotic and salt stress“ has been significanly improved by the authors in accordance with my previous comments. However, I still have some minor comments on the revised manuscript which are provided below.

In Materials and methods, regarding the drought and cold treatments, the authors just gave a reference on their previous study. This is correct; however, I still think that the basic information on the cold and drought treatments whose results are presented in the manuscript has to be given in Materials and methods, i.e., for cold, temperature and length of cold treatment have to be given, and for drought, some characteristics related to soil water content (SWC) and length of the treatment have to be given.

In Abbreviations list, I think that the abbreviations have to be ordered following English alphabet.

Formal comments related to English language and style:

Results, line 172: Add the word „in“ in the statement „3.1. Charcterization of LFBGs in moso bamboo“.

Results, Figure 3 legend, line 262: Correct the typing error in the word „level“ (not „leve“) in the statement „Asterisks indicate the significant level (**p≤0.01) as determiend by Fisher´s Least-Significant-Difference test.“

Discussion, line 382: Add „a“ preecding the word „similar“ in the statement „..indicating PeDFR1 had a similar function to OsDFR.“

Conclusions, line 440: I do not understand the meaning of the statement „Bamboo is an important source of flavonoids with great values…?“ – wheat does it mean „great values“? Maybe it should be modified as follows: „Bamboo is an important source of flavonoids with great economic values….“

Conclusions, line 444: Replace the verb „had“ by „are“ in the statement „There are six types of TF binding sites in the promoters of these DEGs,…“

 

Final recommendation: Accept after a minor revision.

 

Author Response

Dear Editor and Reviewers,

Thank you very much for giving us the opportunity to revise our manuscript entitled ‘Identification of Late Flavonoid Biosynthesis Genes of Moso Bamboo Reveals the Potential Function of PeANR4 Involved in Drought and Salt Stress (manuscript ID: 2442520)’. We have followed you and the reviewer’s suggestions and made revisions carefully. The revised manuscript has been resubmitted. Please check the revised manuscript in details, which is marked in blue.

We hope all these corrections and revisions will be satisfactory and hope that the revised manuscript will be acceptable for the publication in forests.

We thank the reviewers for their concerns and hard work. The responses to the reviewers are listed below.

-----------------------------------------------------------------------------------------

Responds to the Reviewers’ comments:

Reviewer Comments to Author:

The revised manuscript entitled “Identification of late flavonoid biosynthesis genes of moso bamboo reveals the potential function of PeANR4 involved in osmotic and salt stress” has been significanly improved by the authors in accordance with my previous comments.

However, I still have some minor comments on the revised manuscript which are provided below.

 

1. In Materials and methods, regarding the drought and cold treatments, the authors just gave a reference on their previous study. This is correct; however, I still think that the basic information on the cold and drought treatments whose results are presented in the manuscript has to be given in Materials and methods, i.e., for cold, temperature and length of cold treatment have to be given, and for drought, some characteristics related to soil water content (SWC) and length of the treatment have to be given.

Answer: Thank you for your comments and suggestions. We have supplemented it as follows.

 “The separated branches with full young unexpanded leaves were used in drought and cold treatment [28]. The branches were placed on the dry filter paper under room temperature (20 °C and 50% humidity) for drought treatment, and those were put into a chamber set to 0 °C without light for cold treatment, respectively. After 2 h and 8 h, the unexpanded leaves were detached from each treatment and used for RNA-Seq.”

 

Reference:

[28] Huang, Z.; Jin, S.H.; Guo, H.D.; Zhong, X.J.; He, J.; Li, X.; Jiang, M.Y.; Yu, X.F.; Long, H.; Ma, M.D.; et al. Genome-wide identification and characterization of TIFY family genes in moso bamboo (Phyllostachys edulis) and expression profiling analysis under dehydration and cold stresses. Peer J. 2016, 4, e2620.

 

2. In Abbreviations list, I think that the abbreviations have to be ordered following English alphabet.

Answer: Thank you for your comments and suggestions. We have revised it.

 

3. Formal comments related to English language and style:

Results, line 172: Add the word “in” in the statement “3.1. Charcterization of LFBGs in moso bamboo”.

Answer: Thank you for the suggestion. We have supplemented it.

 

Results, Figure 3 legend, line 262: Correct the typing error in the word “level” (not “leve”) in the statement “Asterisks indicate the significant level (**p≤0.01) as determiend by Fisher´s Least-Significant-Difference test.“

Answer: Thank you for the suggestion. We have revised it.

 

Discussion, line 382: Add “a” preecding the word “similar” in the statement ....indicating PeDFR1 had a similar function to OsDFR.

Answer: Thank you for the suggestion. We have supplemented it.

 

Conclusions, line 440: I do not understand the meaning of the statement “Bamboo is an important source of flavonoids with great values…?” – wheat does it mean “great values?” Maybe it should be modified as follows: “Bamboo is an important source of flavonoids with great economic values….”.

Answer: Thank you for the suggestion. We have revised it.

 

Conclusions, line 444: Replace the verb “had” by “are” in the statement „There are six types of TF binding sites in the promoters of these DEGs.…”

Answer: Thank you for the suggestion. We revised it.

Author Response File: Author Response.docx