Transcriptome Profiling Reveals Potential Genes Involved in Salicylic Acid-Induced Arbutin Synthesis in Pear
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe article “Transcriptome Profiling Reveals Potential Genes Involved in Salicylic Acid-induced Arbutin Synthesis in Pear'' is devoted to the important and acute theme of the role of phytohormones in regulation of biosynthesis of phenolic compounds which can be appropriate in medicine care. Authors provide and analyze a lot of information on this theme. The article is well-organized, figures provide enough information illustrating the main conclusions.
I have some comments:
I recommend highlighting hydroquinone glycosyltransferase and species of the pear (Pyrus communis L.?) in the Title.
Introduction should be significantly expanded. Please, concretise the role of arbutin in plant physiology (stress tolerance, for example) and your choice of SA for arbutin biosynthesis induction. What sources of arbutin are important? Is it only pears? What everage content of arbutin in fruits? Please add information on arbutin biosynthesis, in particular, the role of hydroquinone glycosyltransferase in this process.
Line 37: “pharmacological uses for pear”?
Lines 67-76: Give the information on the aim of your investigation.
Mat&met:
Please, add information on the manufacturer (or source) of arbutin (HPLC standard) and hydroquinone.
What concentration of SA was used for RNA-seq?
Results:
Fig. 1-3: Add “The values with different letters indicate significant differences among the treatments containing different concentrations of SA at one point of time at P < 0.05”.
Fig4: Add information on SA-1, Sa-2, CK-1 and CK-2.
Fig. 10 What do the different letters mean? Concretize how significant differences were determined (in the legend).
Author Response
1. I recommend highlighting hydroquinone glycosyltransferase and species of the pear (Pyrus communis L.?) in the Title.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 1-3).
Revised title: Transcriptome Profiling Reveals Potential Genes Involved in Salicylic Acid-induced Arbutin Synthesis in Pear (Pyrus bretschneideri Rehder × Pyrus sinkiangensis Yu)
2. Introduction should be significantly expanded. Please, concretise the role of arbutin in plant physiology (stress tolerance, for example) and your choice of SA for arbutin biosynthesis induction. What sources of arbutin are important? Is it only pears? What everage content of arbutin in fruits? Please add information on arbutin biosynthesis, in particular, the role of hydroquinone glycosyltransferase in this process.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 49-58).
Arbutin was first found in the dried leaves of bearberry (Arctostaphylos uvaursi L.). It also had been identified in pear. Pear, of the family Rosaceae, is the third most widely grown temperate fruit tree species in the world. It plays a key role in people’s daily diet and is important source of arbutin.
3. Line 37: “pharmacological uses for pear”?
In addition, the medicinal functions of pears are constantly being explored.
4. Lines 67-76: Give the information on the aim of your investigation.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 75-81).
In order to explore arbutin content following application of salicylic acid and the bio-synthesis molecular mechanism of arbutin, we firstly investigated the effects of SA on the arbutin content in ‘Yuluxiang’ pear (Pyrus bretschneideri Rehder × Pyrus sinkiangensis Yu) using high performance liquid chromatography (HPLC) and screened differentially expressed (DEGs) associated with arbutin biosynthesis using transcriptome analysis to identify key arbutin biosynthesis genes.
Mat&met:
1. Please, add information on the manufacturer (or source) of arbutin (HPLC standard) and hydroquinone.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 112-113, 130-131).
Hydroquinone (≥98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. It was dissolved in water was added to MS medium to give final hydroquinone concentrations of 220, 440, 660, 880, or 1100 mg L-1.
Arbutin (≥98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd.
2. What concentration of SA was used for RNA-seq?
Answer: Thank you for your suggestion.
100 μM SA treatments of calli was used for RNA-seq and we described it in Lines215-217. In this study, we used tissue culture to ensure that the test material and the environment were more consistent to obtain more reliable results.
Results:
1. 1-3: Add “The values with different letters indicate significant differences among the treatments containing different concentrations of SA at one point of time at P < 0.05”.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 185-187, 195-197, 211-213).
2. Fig4: Add information on SA-1, Sa-2, CK-1 and CK-2.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 240-243)
SA-1 and SA-2 represented two biological replicates of 100 μM SA treatment, CK-1 and CK-2 represented two biological replicates of distilled water treatment. And each biological replicate sample comes from a mixture of callus samples cultivated in three culture dishes.
3. Fig. 10 What do the different letters mean? Concretize how significant differences were determined (in the legend).
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 342-343)
The values with different letters indicate significant differences between Empty vector and overexpression vector at P < 0.05.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript is focused on the identification of arbutin biosynthetic genes by transcriptomic profiling and the elicitation of arbutin production in different Pyrus tissues. Unfortunately, I cannot recommend to publish the manuscript in presented form. I believe that the comments below would be helpful for authors:
Firstly, the scientific name of studied species should be mentioned in the Introduction and Materials and methods.
The Introduction is full of "text-book" information, it should be more fitting to the study, such as missing information about biosynthesis of arbutin. There is no mention about arbutin isoforms, as well. Are the biosynthetic genes known? Why is it important to study biosynthesis of arbutin? The last paragraph should contain the aims of study.
Materials and method:
Two replicates could be insufficient for differential expression analysis. It is unclear how the reads were quantified and the expression levels were analysed (DESEQ package, ...). All the databases and tools should be cited. Which reference genome assembly was used?
Which reporter gene was used during transient transformation (GUS, ...)? How many samles were infected?
What standard was used for HPLC analyses?
Results:
Fig. 4 Does the heatmap represent Z-scores of gene expressions? The color differences are obvious only in the left part.
Figs 9, 10 What is the meaning of letters a, b? The statistical differences between control and treatment?
Supplementary files - several table headers and axes labels are in Chinese.
Comments on the Quality of English LanguageI recommend the grammar check.
Author Response
1. Firstly, the scientific name of studied species should be mentioned in the Introduction and Materials and methods.
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 78, 87-88).
2. The Introduction is full of "text-book" information, it should be more fitting to the study, such as missing information about biosynthesis of arbutin. There is no mention about arbutin isoforms, as well. Are the biosynthetic genes known? Why is it important to study biosynthesis of arbutin? The last paragraph should contain the aims of study.
Answer: Thank you for your suggestion. We have added relevant content in the Introduction (Line 49-58). The study related to biosynthesis of arbutin is very limited, Only hydroquinone glucosyltransferase from Rauvolfia had been identified involvement in the synthesis of arbutin. In recent years, arbutin is believed to have a variety of health benefits for people and plant. Therefore it is important to study biosynthesis of arbutin.
Materials and method:
1. Two replicates could be insufficient for differential expression analysis. It is unclear how the reads were quantified and the expression levels were analysed (DESEQ package, ...). All the databases and tools should be cited. Which reference genome assembly was used?
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 141, 146).
The expression levels were analysed using DESEQ package. The reference genome were downloaded from http://peargenome.njau.edu.cn/allfile/4.Pyrus_bretschneideri_scaffold.gz.
2. Which reporter gene was used during transient transformation (GUS, ...)? How many samles were infected?
Answer: Thank you for your suggestion. The recombined pBWA(V)BS-PbUGT72B1 vector did not contain reporter gene, this study was validated through qRT-PCR. Thirty callus clusters were infected.
3. What standard was used for HPLC analyses?
Answer: Thank you for your suggestion. The standard curve is Y=12.6123129X-10.092234.
Results:
1. Fig4 Does the heatmap represent Z-scores of gene expressions? The color differences are obvious only in the left part.
Answer: The different color in the heatmap represents log10(FPKM+0.000001) of gene expression level.
2. Figs 9, 10 What is the meaning of letters a, b? The statistical differences between control and treatment?
Answer: Thank you for your suggestion. We have revised it in the manuscript (Lines 330-331, 343-344).
3. Supplementary files - several table headers and axes labels are in Chinese.
Answer: Thank you for your suggestion. I have revised it in Table S1 and Figure S4.
4. Comments on the Quality of English Language
Answer: Thank you for your suggestion. The English Language of this manuscript had been edited by International Science Editing (http://www.internationalscienceediting.com).
