Enhanced Catalytic Synthesis of Flavonoid by UV-B Radiation in Artemisia argyi
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The submitted manuscript reports the effect on UV-B radiation on flavonoid production in Artemisia argyi, a study which was conducted by using a transcriptome and metabolome approach. Differentially expressed genes were analyzed, a wide set of differentially accumulated metabolites was identified and classified into ten categories, flavonoids being the largest one. Results correlate with previous studies indicating that the latter accumulate in plants under UV stress and exert a protective effect (for instance, refs. 13-15).
1) In the abstract, a number of 183 differential metabolites is stated (line 16), but the number is 283 in lines 161 and 165.
2) For differentially expressed genes analysis, samples treated with UV-B radiation at 0 h, 4 h, 8 h and 6 d were assayed and in line 82 “a total of 12 libraries” is mentioned. In Material and Methods is stated: “A total of 12 leaf samples, three biological replicates for each time point” (lines 325-326). a) To this reader, “total” is somewhat confusing. If for each time 12 leaf samples + 3 replicates were analysed, I would remove “total”. b) “a total of 12 libraries”: what is meant by library? It would be useful to clear/define it by rewriting lines 325-326.
3) “… “flavonoid biosynthesis”, “flavone and flavonol biosynthesis” … (lines 132, 133, 134, 135 and Figure 2 A, B, C and D): By “flavonoid biosynthesis” is total flavonoid synthesis meant, also including flavones and flavonols? This should be cleared in the text. If this does not comprise flavones and flavonols, “flavonoid biosynthesis” should be relabeled to exclude these compounds.
4) “… only methyl jasmonic acid (methyl-JA)…” (lines 263-264): please correct by: “jasmonic acid methyl ester” (or by “methyl jasmonate”).
5) “Leaves collected at 0 h and 6 d…” (line 343): t = 4 h and 8 h have not been included.
6) Formal remarks
a) Please revise that Artemisia argyi is italicized through the whole manuscript.
b) Please revise carefully that the employed font and size are homogeneous through the whole manuscript. There are many insertions of Times New Roman font (just to give few examples, lines 1, 22, 222, 231, 247, etc) as well as prepositions/articles in bold (for instance, lines 177, 246).
c) Author contributions: In lines 400-402, M.F. (stated four times) does not match with the list of authors (line 4).
Author Response
Comments 1: The submitted manuscript reports the effect on UV-B radiation on flavonoid production in Artemisia argyi, a study which was conducted by using a transcriptome and metabolome approach. Differentially expressed genes were analyzed, a wide set of differentially accumulated metabolites was identified and classified into ten categories, flavonoids being the largest one. Results correlate with previous studies indicating that the latter accumulate in plants under UV stress and exert a protective effect (for instance, refs. 13-15).
1) In the abstract, a number of 183 differential metabolites is stated (line 16), but the number is 283 in lines 161 and 165.
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised accordingly (L16).
Comments 2: For differentially expressed genes analysis, samples treated with UV-B radiation at 0 h, 4 h, 8 h and 6 d were assayed and in line 82 “a total of 12 libraries” is mentioned. In Material and Methods is stated: “A total of 12 leaf samples, three biological replicates for each time point” (lines 325-326). a) To this reader, “total” is somewhat confusing. If for each time 12 leaf samples + 3 replicates were analysed, I would remove “total”. b) “a total of 12 libraries”: what is meant by library? It would be useful to clear/define it by rewriting lines 325-326.
Response 2: Thank you for pointing this out. We agree with this comment. Here, “library” means “cDNA library”. Therefore, we have revised accordingly (L82, L325-326).
“The collected leaf samples were immediately frozen in liquid nitrogen and stored at -80 °C for further analysis.”
Comments 3: “… “flavonoid biosynthesis”, “flavone and flavonol biosynthesis” … (lines 132, 133, 134, 135 and Figure 2 A, B, C and D): By “flavonoid biosynthesis” is total flavonoid synthesis meant, also including flavones and flavonols? This should be cleared in the text. If this does not comprise flavones and flavonols, “flavonoid biosynthesis” should be relabeled to exclude these compounds.
Response 3: Thank you for pointing this out. Here, “flavonoid biosynthesis” refers to the synthesis of total flavonoids, including flavones and flavonols. This has been elucidated in the “Introduction” section (L35-37).
Comments 4: “… only methyl jasmonic acid (methyl-JA)…” (lines 263-264): please correct by: “jasmonic acid methyl ester” (or by “methyl jasmonate”).
Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have revised accordingly (L261-262).
Comments 5: “Leaves collected at 0 h and 6 d…” (line 343): t = 4 h and 8 h have not been included.
Response 5: Thank you for pointing this out. In widely targeted metabolome experiments, we only analyzed samples at 0 h and 6 d, excluding samples at 4 h and 8 h. This is mainly due to the fact that changes in the levels of plant metabolites, especially secondary metabolites, require a long time to be clearly manifested.
Comments 6: Formal remarks
a) Please revise that Artemisia argyi is italicized through the whole manuscript.
Response 6a: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (L3, L97, L147, L366, L368).
b) Please revise carefully that the employed font and size are homogeneous through the whole manuscript. There are many insertions of Times New Roman font (just to give few examples, lines 1, 22, 222, 231, 247, etc) as well as prepositions/articles in bold (for instance, lines 177, 246).
Response 6b: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly.
c) Author contributions: In lines 400-402, M.F. (stated four times) does not match with the list of authors (line 4).
Response 6c: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (L401-403).
Reviewer 2 Report
Comments and Suggestions for Authors
Questions and comments are as follows
1.The Authors described many enzymes under their trivial names (in fig. 6, for example) and abbreviations PAL, 4CL, C4H, CHI (PAL, 4CL, C4H, CHI). It is recommended to indicate the EС classification numbers of the enzymes if these numbers are known.
2. The Reviewer found a description of only one direct analytical method, namely spectrophotometric (OD at 470 nm), performed with calibration using rutin as standard. Since many metabolites have been described, what analytical methods (spectroscopy, chromatography, electrophorese) were used (except the Database and calculations results)?
3. It is recommended to change “6 days” to “144 hours” for uniformity of presentation and clarity. What is the reason for choosing six days as the final experimental time??
4. What is the intensity of UV irradiation? What is the difference in the spectra of natural UV radiation from artificial UV-B? What source of artificial UV-B irradiation was used for laboratory experiments?
The dimensions of UV-B radiation and light intensity are indicated as “μmol m−2 s−1”. Check and explain what “µmol” mean.
4. How was the effect of UV radiation on plants (growth rate, biomass gain, etc.) experimentally measured?
5. It is recommended to provide a list of the most frequently used abbreviations, for example, such as DEG, DAM, UV-B.
6. Section 4 “Conclusions” is recommended to be supplemented with specific experimental results described in Abstract.
7. The authors “predicted the critical importance of flavonoids for the adaptation of A. argyi to UV radiation”, but did not specify the reasons for this prediction. One of the experimental results is an increase in flavonoid content in A. argyi grown at different altitudes, namely, an increase of 45%. On the other hand, any quantitative information about other metabolites is completely absent. Is the increase in flavonoid content common to all plants grown at high altitudes or only to A. argyi?
Typos such as a different font are highlighted in yellow color in the pdf-file.
Comments for author File: 
Comments.pdf
Author Response
Comments 1: The Authors described many enzymes under their trivial names (in fig. 6, for example) and abbreviations PAL, 4CL, C4H, CHI (PAL, 4CL, C4H, CHI). It is recommended to indicate the EС classification numbers of the enzymes if these numbers are known.
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (P2, L53-58; P8, L227-234; P9, L269-273).
Comments 2: The Reviewer found a description of only one direct analytical method, namely spectrophotometric (OD at 470 nm), performed with calibration using rutin as standard. Since many metabolites have been described, what analytical methods (spectroscopy, chromatography, electrophorese) were used (except the Database and calculations results)?
Response 2: Thank you for pointing this out. UPLC-MS was used in the widely targeted metabolome analysis conducted by Metware Biotechnology Corporation. Due to the focus of this study on the synthesis of flavonoids, other substances mentioned in the manuscript were not further validated.
Comments 3: It is recommended to change “6 days” to “144 hours” for uniformity of presentation and clarity. What is the reason for choosing six days as the final experimental time??
Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have changed “6 days” to “144 hours” in the text and charts of the manuscript.
Before collecting experimental materials for metabolome analysis, we tested the total flavonoid content and found that the accumulation of total flavonoids reached its peak at 6 days. Therefore, we chose 6 days as the final experimental time.
Comments 4: What is the intensity of UV irradiation? What is the difference in the spectra of natural UV radiation from artificial UV-B? What source of artificial UV-B irradiation was used for laboratory experiments? The dimensions of UV-B radiation and light intensity are indicated as “μmol m−2 s−1”. Check and explain what “µmol” mean.
Response 4: Thank you for pointing this out. The intensity of UV-B irradiation used in our experiment is 2 μmol m−2 s−1. The spectra range of natural UV radiation is about 200～400 nm. According to the wavelength range of UV radiation, it is divided into three bands: UV-A (320～400 nm), UV-B (280～320 nm), and UV-C (200～280 nm). In this study, we selected UV-B (295 nm), provided by Huizhou Kedao Technology Co., Ltd, as the light source. Light intensity unit “μmol m−2 s−1” means the photon flux density per unit time (s) on a unit area(m2). The unit of photon flux density is “µmol”.
Comments 5: How was the effect of UV radiation on plants (growth rate, biomass gain, etc.) experimentally measured?
Response 5: Thank you for pointing this out. The effects of UV radiation on plants include growth, physiological and biochemical response, etc. For example, the hypocotyl length of seedlings, plant height, leaf area, root length, and number of branches of plant can be detected to determine the growth status of plants. Photosynthetic rate, transpiration rate, and stomatal conductance can be measured to indicate physiological and biochemical status of plants.
Comments 6: It is recommended to provide a list of the most frequently used abbreviations, for example, such as DEG, DAM, UV-B.
Response 6: Thank you for pointing this out. Though there are many abbreviations in the manuscript, the most frequently used abbreviations are not numerous. These abbreviations, such as DEG, DAM and UV-B, are also well-known in this field. Such, the authors believe that these abbreviations do not affect the reading of interested readers.
Comments 7: Section 4 “Conclusions” is recommended to be supplemented with specific experimental results described in Abstract.
Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the section “Conclusions” and “Abstract” accordingly.
Comments 8: The authors “predicted the critical importance of flavonoids for the adaptation of A. argyi to UV radiation”, but did not specify the reasons for this prediction. One of the experimental results is an increase in flavonoid content in A. argyi grown at different altitudes, namely, an increase of 45%. On the other hand, any quantitative information about other metabolites is completely absent. Is the increase in flavonoid content common to all plants grown at high altitudes or only to A. argyi?
Response 8: Thank you for pointing this out. The critical importance of flavonoids for the adaptation of A. argyi to UV radiation was proposed by the transcriptome and metabolome analysis. Due to the strong antioxidant capacity of flavonoids, flavonoids may help alleviate the elevated levels of reactive oxygen species (ROS) in cells caused by ultraviolet radiation. This has been explained in the manuscript (P6, L191-194).
By DEGs and DAMs enrichment analysis, flavonoid biosynthesis was believed to play a key role in the adaptation of A. argyi to UV radiation. So, the authors focused on the analysis of flavonoids rather than all substances. Accordingly, no quantitative information on other metabolites was presented in the manuscript.
The results indicated that A. argyi planted in high-altitude areas with increased UV exposure accumulated higher levels of flavonoids. Further research is needed to determine whether this phenomenon exists in other plants.
Reviewer 3 Report
Comments and Suggestions for Authors
Suggestions and comments for Authors are attached
Comments for author File: Comments.pdf
Comments on the Quality of English Language
Extensive editing of English language required through out the submitted manuscript
Author Response
Comments 1: In this manuscript “Enhanced catalytic synthesis of flavonoid by UV-B radiation in Artemisia argyi”, the authors have comprehensively studied the effect of stress through UV-B radiation on enhanced catalytic synthesis of secondary metabolites like flavonoids in Artemisia argyi. The authors conducted a thorough study however manuscript lacks the co-relation between the findings and investigated objectives. It needs to be addressed carefully. However, reviewer considers that overall, the article could be published in catalysis and is of interest for the readers, but major revisions are required, summarized as follow:
The abstract needs to be modified as it must reflect both experimental and computational studies and outcomes. Same recommendation for the conclusion part.
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the section “Conclusions” and “Abstract” accordingly.
Comments 2: In line 31, These active constituents not -------- use combating disease. names the diseases and add more references.
Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly.
“These active constituents not only protect plants from biotic and abiotic stresses, but also serve as anthelmintic, antibacterial, antirheumatic, and antispasmodic agent for the treatment of diseases such as malaria and hepatitis [4−6].”
Comments 3: In line 39, one or two lines to clear the term auxin transport to make it more informative for general readers.
Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 48).
Comments 4: Line 39-49, not clear and confusing rewrites these lines.
Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly.
“Flavonoids possess multiple physiological functions in plants. As antioxidants, they can resist ultraviolet (UV) damage and oxidative stress through several mechanisms: (i) they can chelate transition metal ions, thus reducing the level of reactive oxygen species; (ii) they can stabilize unpaired electrons; (iii) they are highly reactive as hydrogen or electron donors [9]. Anthocyanins can accumulate in plant cells under UV stress and are believed to play a key role in UV radiation resistance [10−11]. In most cases, the anabolism of flavonoids in plants is inhibited by biotic stresses such as pathogens and pests. However, some flavonoid metabolites, like catechins, quercetin, and rutin, are synthesized in large quantities to combat pathogens and pests [12−14]. In addition, flavonoids promote the accumulation of auxin by inhibiting polar auxin transport under stress, thereby regulating plant growth and enhancing stress resistance [15, 16].”
Comments 5: Line 54, cinnamate4-hydrox-53 ylase (C4H) and 4-coumarate-CoA ligase (4CL)??, ---need to add reference.
Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 52).
Comments 6: Line 62 --- methyl flavonoids, respectively?? And line 67----- flavonoid synthesis pathway?? Add references.
Response 6: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 63 and 68).
Comments 7: Lines 73-77 need to rewrite its meaning are not clear.
Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 70-74).
Comments 8: Lines 80-110, need to be written little bit in less technical language to make it more readable for other fields.
Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 82-83).
Comments 9: In running text (table S1) and (table S2) lines 84, 88, where these tables are in submitted manuscript.
Response 9: Thank you for pointing this out. Table S1 and table S2 were attached in the supplemental file (Page 4 and Page 5).
Comments 10: In section 2.2, line 114, page 3, “By harnessing bioinformatic databases such as GO and KEGG, the most relevant biological pathways for the DEGs were obtained”, Cite the proper reference. Also, any specific reason to consider GO and KEGG for the present study? And how did you locate the relevant data?
Response 10: Thank you for pointing this out. Harnessing bioinformatic databases such as GO and KEGG to cluster differentially expressed genes (DEGs) and annotate their metabolic pathways can help interpret gene functions. These bioinformatic databases are widely used in transcriptome analysis. In this study, cDNA sequencing and data analysis were performed by Metware Bio-technology Corporation (Wuhan, China).
Comments 11: How the accuracy of the data obtained from KEGG and GO were assessed?
Response 11: Thank you for pointing this out. KEGG and GO are the international standard classification systems for gene function, applicable to various species. The accuracy of the data obtained from KEGG and GO is generally further verified through metabolomics analysis or other experiments.
Comments 12: The authors have not discussed the comparative analysis of the data obtained from KEGG and GO.
Response 12: Thank you for pointing this out. The expression of genes varies significantly among different species and under different treatment conditions, resulting in significant differences in the results of GO and KEGG analysis. This makes it difficult to compare and discuss these results. Due to the lack of relevant reports, the authors have not discussed the comparative analysis of the data obtained from KEGG and GO.
Comments 13: In section 2.2, lines (121-124), page 3, “Furthermore, “phenylpropanoid biosynthesis”, “alphalinolenic acid metabolism”, “stilbenoid, diarylheptanoid, and gingerol biosynthesis”, “phenylalanine, tyrosine, and tryptophan biosynthesis”, and “phenylalanine metabolism” were highly enriched in the UV4h and UV8h DEGs, suggesting that these metabolic pathways might be conducive to the early adaption of A. argyi to UV radiation”
How did you draw this conclusion? Supporting references are also missing.
Response 13: Thank you for pointing this out. Since “phenylpropanoid biosynthesis”, “phenylalanine, tyrosine, and tryptophan biosynthesis”, and “phenylalanine metabolism” are upstream pathways of flavonoids, the enrichment of DEGs in these pathways implies significant changes in the synthesis of flavonoids. “alphalinolenic acid metabolism”, “stilbenoid, diarylheptanoid, and gingerol biosynthesis”are also related to the resistance of plants to UV radiation or other stress (Saradhi PP, Alia, Arora S, Prasad KV. Proline accumulates in plants exposed to UV radiation and protects them against UV induced peroxidation. Biochem Biophys Res. Commun. 1995, 209(1):1-5. Zhou J, Huang J, Tian X, Zheng J, He X. Transcriptome analysis reveals dynamic changes in the salt stress response in Salix. J. Forestry Res. 2020, 31(5):12.). The authors have added supporting references in the manuscript (Line 124).
Comments 14: In section 2.2, lines 137-138, page 2, “Hence, it can be inferred that the DEGs involved in the
“flavonoid biosynthesis” and “flavone and flavonol biosynthesis” might be more closely related to the resistance of A. argyi against UV radiation.” Add proper references. Also how did you conclude that statement?
Response 14: Thank you for pointing this out. Since the great majority of DEGs enriched in “flavonoid biosynthesis” and “flavone and flavonol biosynthesis” pathways were upregulated, the authors inferred that “flavonoid biosynthesis” and “flavone and flavonol biosynthesis” might be more closely related to the resistance of A. argyi against UV radiation. We have added supporting references in the manuscript (Line 138).
Comments 15: On page 5, line 163, the findings from Heat map must be elaborated in detail along with supporting references from literature. Also, captions should contain details regarding the figure so that one may understand the contents of figure without looking at the text in discussion.
Response 15: Thank you for pointing this out. The differential metabolites of all comparison groups were collected and merged as a differential metabolite set for hierarchical clustering analysis. Z-score was used to standardize the data and a clustering heatmap was drawn for each differential group. In this study, metabolomics analysis was performed by Metware Bio-technology Corporation. The heatmap of DAMs is widely used in metabolomics analysis. We have added captions in the manuscript accordingly (Line 173-175).
Comments 16: On page 5, section 2.4, first para, line 177, how the outcomes of largest group of DAMs are inconsistent with your transcriptome analysis? must add references.
Response 16: Thank you for pointing this out. In the manuscript (line 177), “The largest group of DAMs identified by KEGG enrichment analysis was “bio-synthesis of secondary metabolites”, which is consistent with our transcriptome analysis (Figure 2A, B, and C).” There is no “inconsistent” in this section.
Comments 17: Also cite references to the statement (line 275, section 2.6, page 9), lines 311-314 on page 10, all the databases mentioned in material and method section. Also cite the references of the software used during this study.
Response 17: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 279). Lines 311-314: “Although we cannot rule out the contribution of other factors that may differ between the sites, such as low temperature or exposure to biotic stress, it seems likely that this difference can at least be partly accounted for by the increased levels of UV exposure at high altitude.” It is well known that ultraviolet radiation is enhanced in high-altitude areas. Such, we inferred that this difference can at least be partly accounted for by the increased levels of UV based on the results of multi-omics analysis and this fact. We have checked the manuscript accordingly.
Comments 18: Correct the formatting of line 341, page 11
Response 18: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript accordingly (Line 343).
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I appreciate the careful revision of the manuscript which has been done by the authors.
Reviewer 3 Report
Comments and Suggestions for Authors
The authors have incorporated all suggested changes in submitted article. I have no comments.
Comments on the Quality of English Language
Little bit need to improve
