Integrated Volatile Metabolomics and Transcriptomics Analyses Reveal the Influence of Infection TuMV to Volatile Organic Compounds in Brassica rapa

Round 1

Reviewer 1 Report

horticulturae-1506819

The authors aim to analize the volatile organic compounds composition in Brassica rapa as influenced by the infection of Turnip mosaic virus. The integrated use of metabolonic and transcritpion analyses is a very intetersting integration to the traditional analyses of the influence of stresses (in this case a virus) on the Vocs composition.

The introduction clearly describes the virus, its distribution and the problems related to its infection. The role of Vocs in the spreading of the infection and its vectors are described in a complete way.

Material and methods are very well written, with plenty of information that permit any other researcher to replicate all experiments.

Results are presented in a clear way with plenty of figures and tables which make the results veru friendly available and understandable to the reader. Namely Fig. 7 is a very nice example of how to disseminate scientific knowledge.

The discussion is well balanced between the results obtained by the authors and those found in the literature.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper deals with the use of transcriptomics and metabolomics to study how the infection of turnip mosaic virus modifies the volatile organic compound that Brassica rapa releases. The authors inoculated a TuMV-susceptible and a TuMV-resistant genotype and compared their leaves, measuring transcripts by RNA-Seq and volatile metabolites by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). They found interesting transcriptomic differences between the four groups (inoculated and non-inoculated, susceptible and resistant) and genes clustering together for their differential expression between the four groups.They cleaned and checked volatile metabolomics data by well-established procedures of quality control (visualization of TIC, PCA, Clustering), found 20 metabolites differentially accumulated. In order to integrate transcriptomic and volatile metabolomic data, correlations were calculated and hypotheses were proposed for the influence of TuMV inoculation on release of volatile metabolites.

The paper contains many details and tries to make the most of all the data by relate them together and propose hypotheses. However the level of english language of this paper has to be improved.

 

Main comment:

The comparison of only two genotypes cannot identify for sure the metabolites behind the difference of response, because these differentially accumulated metabolites could be caused by any other genome differences. A safer way to identify these metabolites would be to test several susceptible and several resistant genotypes, and pinpoint the common differentially accumulated metabolites.

 

Major comments:

L253-254: please do not write « repeatability was good », as the Heatmap and Z-score plot in fig S3 seem to indicate a high heterogeneity in the measurements.  You’d rather assess the repeatability with calculated numbers (RSD).

L337-341 (and fig 9A): The reader is not aware how pathways enrichment are calculated for volatile metabolites. Was it based on the ratio of number of differentially accumulated metabolites in this pathway related to number of detected metabolites in this pathway? Or on the number of differentially accumulated metabolites in this pathway related to number of theoretical metabolites in this pathway? Because the detection method itself can bias this analysis a lot towards pathways that contain many volatiles, if you consider only the 2^nd calculation method. Please detail your method of enrichment calculation for metabolites.

 

Minor comments:

Please, all along the paper, do not write « metabolome » but « volatile metabolome » : what you deal with is only a part of the metabolome and contains only 99 compounds.

L316: « Cellular processes were the least enriched ». Is it really relevant ?

L319: please do not jump too fast to conclusions by writing « TuMV stress could accelerate the increase of VOCs »

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Minor comments:

• It is desirable to indicate more clearly the Conclusion section.
• Change Reference list according to the Journal’s rules (journal titles and journal volume should be in Italics; year of publication- use bold letters)
• Don’t use abbreviation of ‘Figure’ in the text, write ’Figure’ instead of ‘Fig.’
• Not all e-mails are included in the title page
• Postal indexes are absent on the title page
• Line 69-70: ‘The composition and content of volatiles released by herbivorous insects change significantly after plants are attacked by herbivorous insects’.- suppose that this is a misprint and ‘released by herbivorous insects’ should be replaced by ‘released by plants’
• Line 109 Repetition: ‘The TuMV homogenate was gently wiped on the leaves in the direction of the vein’s direction’
• Line 184 ‘from the ELISA methods, the resistant index was divided into four ranks, i.e., highly resistant (P/N≤1), highly susceptible (P/N>4) .- only two ranks are indicated, but not four
• Line 230 :’ the detected metabolites was combined’ change to ‘the detected metabolites were combined’
• Figure 3d- sharpen the letters- otherwise it is difficult to read the data
• Please use bold letters in Figures 4a,b- it is difficult to see the details
• Lines 343-344- repetition: ‘Pearson’s correction coefficient (PCC) analysis showed that of the genes and metabolites with inconsistent regulatory trends, metabolites were upregulated’
• Line 405 ‘to found out’ change to ‘to find out’
• Line 460 ‘By metabolome, what is the final differential metabolites’ change to ‘By metabolome, what are the final differential metabolites’
• Line 568 ref.40: use italics for Brassica napus
• Check abbreviations of the journals in the Reference list:
• Ref 32 use abbreviation of the journal’s title: Rev. Entomol.;
• the same for 31: Ecol Lett.
• And Ref 33 :use Curr.Opin. Virol. Abbreviation.
• The same for references 30, 29, 27, 16,11 10 etc….

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thanks to all the answers and improvements that were given after the reviewing, this paper seems to me to be fit to print.