Accumulation of Polyphenolics and Differential Expression of Genes Related to Shikimate Pathway during Fruit Development and Maturation of Chinese Olive (Canarium album)
, Naiyu Wang 1,2, Junyue Zhao 1,2, Yan Zhao 1,2, Rong Xu 1,2, Fanghao Fu 1,2, Tengfei Pan 1,2, Yuan Yu 1,2,3, Zhixiong Guo 1,2,* and Wenqin She 1,2,*
Round 1
Reviewer 1 Report
The article titled "Differential expression of genes related to shikimate metabolism during fruit development and maturation of Chinese olive (Canarium album)" tracked the changes in phenolic compounds and gene expression of selected genes coding for enzymes involved in shikimate metabolism in three Chinese olive cultivars from 50th DAF to 170th DAF. This manuscript could not be recommended for publishing in Agronomy in its present form. It requires a serious audit of the Results and Conclusion sections.
I would like to suggest the authors consider changing the manuscript’s title. As it focuses on phenolic composition and gene expression analysis it is unclear why the authors decided to point out “differential expression”.
The conclusion section is missing information about the potential use of results obtained in this study. What would be the author’s idea for future research? “DAHPS-1 and CMs were key genes that affect the difference of polyphenol synthesis in different Chinese olive cultivars” – please explain.
All other comments and suggestions are given in the pdf document.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Summary
The work outlines the differences in phenolic compounds and their precursors in three Chinese olive cultivars. The paper is very descriptive describing the level of change of metabolites and some correlation between composition and shikimate gene expression over fruit development. The new knowledge reports on the differences in shikimate metabolism and the relationship to polyphenol composition in three Chinese olive cultivars.
Concept Comments and Major corrections required
I would suggest a few major modifications to the paper.
Firstly, the introduction would benefit from the question/ problem that the research addresses being more clearly defined and woven into the “story”. A clearer introduction regarding why understanding the shikimate pathway in the olive is important and what its benefit are. Woven in with the description of what the Shikimate pathway does would make for better reading and setting of the “scene”. It is very heavy going, making the reader question why the authors are explaining all this detail of a metabolic pathway.
Secondly, the results would benefit from being analysed differently to show where there is a significant trend over time for a cultivar and then between those trends are there significant differences in trend for the different cultivars. Instead of the sampling-to-sampling differences that are laboriously described using terms as higher and less which have no definitive context.
Lastly, with the results clearly highlighting the significant differences in the metabolites and the associated gene expression. It would then create a more substantial base for the discussion. Enabling a clear informative discussion to flow from the results and better highlighting the significant outcomes of this work.
Minor corrections required
Lines 20-31 Abbreviations need to be written in full as this needs to be standalone. i.e Tx, Cy, LF and DAHPS-1, CM2, CMs
Line 39 What is ‘mainly responsible?’
Line 77 Abbreviation AtCM1 in full
Line 78 AtCM2 in full
Line 79 PhCM2 in full
Line 95-95 ‘Regulatory mechanism’ of what
Line 103 ‘realted’ should be related?
Line 124 ‘described by’ what/who?
Line 126-138 Method clarification required. Line 126 I would suggest you outline here that the supernatant is decanted and reserved. Line132-133 says that TFA was added to the solvents. Does that mean all solvents from line 126 onwards? If so, then state that the methanol was 0.01% TFA there.
Line 141 ‘described by’ what/who?
Line 167 Which fruits? How many? Was it a subsample weighed out of the fresh ground tissue used for the 2.2.1?
Lines 191-336 What defines a low level or a higher level? Lower than other varies or fruit in general? It is better to say that the content remained steady until X DAF when the amount increased x fold. This can be said for the entire results section. Hence the suggestion that you analyse the data differently by assessing whether there is a significant change within a cultivar over the growing period. Then comparing the cultivars.
Figure 2 What are the error bars?
Lines 270-272 The decline in expression is this significant between the sampling times. Is Cy being higher significant?
Figure 3 What are the error bars?
Lines 289-290 ‘Decreasing range’. Do you mean, a significant decrease in expression?
Figure 4 What are the error bars?
Lines 377-378 ‘High level of quinate accumulated in the fruit contributes to the tart and astringent flavor of Chinese olive’ who says?
Line 390 ‘Gallate synthesised’? Are you meaning the gallate that was synthesized?
Line 391 ‘High contents of conjugated gallate and ellagate were detected throughtout
the development and maturation of Chinese olive implying biosynthesis of gallate was
active in the fruit.’ High compared to what? How can you make this statement without putting it in context or defining what high is?
Lines 402-403 ‘DAHPS-1 expressions were closely related to the accumulation of flavonoids such as quercetion derivative and kaempferol derivative among the three Chinese olive fruits’. I do not see these compounds in the papers results are you referring to another paper?
Author Response
请参阅附件。
Author Response File: Author Response.pdf
Reviewer 3 Report
This manuscript provides insight the relationship between shikimate metabolism and phenolic compounds biosynthesis. Three Chinese olive fruits (cv. TX, CY and LF) of distinct flavor were utilized as materials, and large differences were discovered in phenolic compounds contents and the expression of the genes related to the shikimate pathway among the three cultivars.
The topic is original and relevant in the field, and it addresses a specific gap in the field. The references are appropriate.
The paper is well written. However, I strongly suggest providing a mechanistic illustration summarizing all of the components studied in this experiment.
Conclusion section:
Should be improved; we note the absence of a clear general conclusion that identifies the perspectives that can follow up on this work
Resolution of figures needs to improvements.
Author Response
请参阅附件。
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors have revised the manuscript, changed its title to a more suitable form, and changed the statistical analysis according to the given instructions. However, the authors have not made all the corrections given in the previous round of review (points: 5, 11, 15, 16, 18, and 19 in the cover letter) so I could not recommend this article for publishing.
Author Response
Response to Reviewer 1 Comments
Point 1: Lines47 “[6]” - Please check if this is the correct reference
Response 1: We have modified this reference as follows :” Shikimate pathway can transform more than 20% of fixed carbon into aromatic compounds, which are precursors for the synthesis of phenols, flavonoids, indole hormones, lignin and and other secondary metabolites in plants[6]”
Point 2: Lines208-211 “(ios)corilagin” - ios or iso?.
Response 2: We have changed all " ios " to " iso " in the paper.
Point 3: Lines267 “The expression level of LF was the lowest on 50th DAF,” - Is this correct? According tho figure 3B the lowest level of DAHPS-2 in LF cultivar was recorded 70 days after anthesis. Please check.
Response 3: The highest expressions of DAHPS-2 among the three cultivars all appeared on 50th DAF. We want to express is that the highest expression of LF was lower than TX and CY.
We have modified to “The highest expressions of DAHPS-2 in TX, CY and LF all appeared on 50th DAF (Figure 3B). The highest expression of LF was 62.50% of TX and 55.16% of CY (Figure 3B).”
Point 4: Lines271-272 “The DAHPS-3 expressions in the TX and LF were the highest on 170th DAF,” - Please check. Is DAHPS-3 expression in LF cultivar highest in 170 DAF or 90 DAF?
Response 4: We have modified some errors in the statistical analysis of Figure 3C. We have revised this sentence in the paper to “The highest expression of DAHPS-3 in TX appeared on 170th DAF, and which in CY and LF both appeared on 50th DAF(Figure 3C).” .
Point 5: Lines372-373 “Shikimate accumulated relatively less compared to quinate during Chinese olive fruit development.” - Please check. According to Figure 2 shikimat content gradually increase in 150 and 170 days after anthesis whereas quinate content remains relatively stable. On the other hand quinate is more abundant in all measured time points than shikimate.
Response 5: We have modified to “Quinate was more abundant in all measured time points than shikimate whose content was less than 0.2% of quinate content.”
Point 19: Lines422-444 “In additon, the results of the fusion expression with GFP demonstrated that, the CM-1 and CM-2 of Chinese olive were localized as expected into the chloroplast/plastid and cytosol, respectively[40].” - It is unclear how is this connected to expression levels of CMs in different cultivars.
Response 19: We have added and modified as “In additon, the results of the fusion expression with GFP demonstrated that, the CM-1 and CM-2 of Chinese olive were localized as expected into the chloroplast/plastid and cytosol, respectively[40]. Cytosolic Vitis vinifera chorismate mutase gene (VvCM) was positively correlated with polyphenols and flavonoids. The enzyme encoded by this gene was not regulated by metabolites (Phe, Tyr, Trp), while plastidial VvCM could be activated by Trp[43]. Cytosolic At CM2 provided essential substrate Phe for Arabidopsis under stress conditions[20]. The expression of cytosolic CM-2 was predominant, bearing 50~ 500 folds of the plastidial CM-1, throughout the whole stage of fruit development and maturation, while the CM-1 was up-regulated and the predominated CM-2 was down-regulated, suggesting that they may play different roles in regulating the biosynthesis of aromatic amino acid and secondary phenylpropanoid metabolism of Chinese olive.” .
Reviewer 2 Report
The points raised have been addressed. The conclusion is much tighter.
Author Response
Response to Reviewer 2 Comments
Thanks for your kindly advises. All the changes were made using revision mode. Please review it again. Thank you for your attention to our article.
