Transcriptome and Co-Expression Network Analyses Identify the Molecular Signatures Underlying Drought Resistance in Yellowhorn

Round 1

Reviewer 1 Report

Liu, et al., have presented a transcriptome analysis of several different accessions of a commercially useful tree called yellowhorn (Xanthoceras sorbifolium).  For this analysis, leaves were harvested from 10 individuals after 6 months of severe drought.  They then prepared and sequenced RNA from these plants and did extensive bioinformatic analysis of the dataset in order to identify specific functional classes and co-varying gene sets that might link growth rate and water consumption with adaptations.  In the end, they were able to identify several broad physiological processes likely to contribute to the differences (”nitrogen, proteins and cellwall components biosynthetic and metabolic process as key pathway involved in WUE regulation while sugar,microtubule and phosphatidylinositol biological and metabolic process as key pathway involved in growth regulation. IAA biosyntheticand metabolic process may regulate the drought tolerance”), but I am concerned that the majority, if not all, of these could have been predicted before the study began.

I am well aware of the work that went into this project, and would not like to see it discarded, but I think some changes need to be made to make the data more accessible to naïve readers, and perhaps to clarify the limitations of the experimental design. 

The following are some places the authors might consider making changes:

1. I think the first major stumbling block for naïve readers such as myself was the English grammar.  I started correcting it on my copy, but began running into phrases such as one on line 77, “in the middle of the same direction”, that I did not understand, and so could not correct.  Others were more common problems such as the use or absence of “the” (the hardest word in the English language according to the BBC) and plurals singular. I  would strongly encourage the authors to have a language specialist go through the document and correct it to a common standard.  It is possible that if these things didn’t distract me (as a reviewer), I would have been able to follow the arguments more closely;
2. The second block, that perhaps only affects run of the mill biologists such as myself, was the reference to modules as color blocks (e.g., lines 23 and then 181 onward). I have not seen this before, despite having read and written several transcriptome papers.  I think it deserves some explanation (are these colors standard for specific kinds of networks, or only being used to distinguish the modules in this paper?  If the latter, I think the modules need different names even if some people would argue those names bias the interpretation of the network within them.).  I think it is also appropriate to give the tables in S2 a short legend telling the reader what to learn/conclude from them, and especially, why some text or text background was in red, some in grey, some in green, and many in black and white.
3. Several other aspects of the work bothered me, but would be much harder to correct than those above. Nevertheless, if the authors could address any of them, I think the paper could be improved.
4. It would be helpful if the authors drew horizontal lines in Table S1 to separate the 4 classes of plants;
5. It seemed that some of the samples in S1 were meant to be duplicates within each of these classes. Could the authors do a PCA analysis to show how the data sets clustered with each other;
6. They were working with field-grown plants. Could some of the gene variation seen be due to microenvironmental differences between the plants?  It would have been nice to have sampled the same trees 2 or more times over a given period to see if the expression levels were reproducible.  It might have also been nice to have watered the plants after the drought and then determined which genes were differentially expressed due to the genotype and which to drought (acknowledging there is an important interaction between the two parameters);
7. Faced with 2000+ DEGs, I personally would have increased the stringency to log2³2 or higher so that the discussion could focus on where the biggest differences in DEGs lay. All of these cut-off points are arbitrary.  Once one acknowledges that one can never know all DEGs in any response, or which magnitudes of difference have biological consequences, you can justify why you are only analyzing the most dramatically affected fraction of the DEGs;
8. It would have been useful to confirm the changes that were seen by semiquantitative PCR on a few of the genes using independent RNA collections from the same trees or ones classified in the same way. This would demonstrate the value of this analysis;
9. On line 54, the authors write “Moreover, the study of the physiological characteristics of yellowhorn seedlings revealsthat the content of osmotically regulated substances (soluble sugar, soluble protein and proline) and anti- oxidativeenzymes (SOD, POD and CAT) activity were increased by drought stress [5,14]”. Perhaps the authors could look through their data and determine which specific genes in these responses (as well as those involved in trehalose and sucrose metabolism; mentioned on line 246) are differentially expressed between their different trees, and to what magnitude, and then discuss the meaning of that agreement or disagreement.  I note that the authors did discuss KEGG-defined classes of processes, but I didn’t find the conclusions very clear; I have become disenchanted with the overly generalized groupings shown in Figs 2 and 5. I also don’t benefit from discussions that focus on “DEGs” as if genes showing increases and genes showing decreases in expression can be grouped together.  It would be interesting if the authors addressed specifically which pathways (not processes) were induced, and which suppressed.

I realize I have focused on the negative aspects of the paper, as I see them, and for that I apologize.  But I think addressing any of these points would open the paper to more readers.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The review of manuscript titled “Transcriptome and Co-Expression Network Analyses Identify the Molecular Signatures Underlying Drought Resistance in Yellowhorn" by Xiaojuan Liu, Yifan Cui, Zhiyan Wu, Yang Zhao, Xiaoyu Hu, Quanxin Bi, Suzhi Yang, LibingWang

The manuscript by LibingWang (corresponding author) and others aimed at investigating the

transcriptome analysis of four types of yellowhorn differing in drought resistance and growing under the long-term drought conditions. The authors used RNA library construction and sequencing and obtained two groups of differentially expressed genes in water-saving/ water-consuming and fast-growing/ slow-growing yellowhorns. The results of the experiment are interesting and touch on the molecular basis of yellowhorn adaptability to drought conditions. The manuscript is well-written; however, it contains deficiencies which, in my opinion, require attention.

 Abstract and Introduction

The Abstract section of the manuscript is well written, and it contains essential results and the most important conclusions. The Introduction section contains the most important and actual state of knowledge in the manuscript subject. The authors show the important aspects of controlling plant metabolism in drought conditions including physiological, cellular and molecular processes (Lines 38-50). They also showed the factors affecting the yellowhorn response to drought (Lines 56-59). The main problem is that the analysis lack details about expression of genes involved in response to oxidative stress, which is one of the important effects of drought stress. It would be very beneficial for the molecular picture presented in the manuscript to characterize genes associated with protection against oxidative stress. These genes play not only a protective role against this stress but also a regulatory role and control the expression of many genes involved in specific metabolic pathways. It should be completed not only in the Introduction section of the manuscript but also in Results and Discussion.

 Materials and Methods

Thus section is well written and in my opinion, contains all necessary information.

Results and Discussion

I think that Table 1 (Line 143) should be placed in supplementary data  

Line 155-156: The authors mention regulatory, control, and long-term plant responses to drought stress. For this reason, the discussion should include a fragment concerning both the direct effects of oxidative stress resulting from drought stress and the effects of the increase in the intensity of ROS formation and its consequences for the control of cell and organ metabolism.

Line 170 (Table 2) and line 206 (Table 3): limit the number of decimal places

As mentioned earlier, among the sub-chapters in the Discussion section, there is no part on the control of gene expression involved in protection against oxidative stress. On the other hand, the authors included other types of controls at different molecular levels, such as phosphorylation / dephosphorylation of proteins.

References:

Line 354: Xanthoceras sorbifolium change to Xanthoceras sorbifolium (italics)

Line 357: Xanthoceras sorbifolium change to Xanthoceras sorbifolium (italics)

Line 360: Xanthoceras sorbifolium change to Xanthoceras sorbifolium (italics)

Line 363: Xanthoceras sorbifolium change to Xanthoceras sorbifolium (italics)

Line 366: Xanthoceras Sorbifolia change to Xanthoceras sorbifolia (italics)

Line 373: Leucaena leucocephala change to Leucaena leucocephala  (italics)

Line 384: Xanthoceras Sorbifolia change to Xanthoceras sorbifolia (italics)

Line 389: Picea glauca change to Picea glauca (italics)

Line 399: Glycine max change to Glycine max (italics)

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have attempted to address several of the issues raised in the previous review.  I have added my thoughts on some of these but do not expect the authors to address them.

 Response to comments

Reviewer #1

Liu, et al., have presented a transcriptome analysis of several different accessions of a commercially useful tree called yellowhorn (Xanthoceras sorbifolium).  For this analysis, leaves were harvested from 10 individuals after 6 months of severe drought.  They then prepared and sequenced RNA from these plants and did extensive bioinformatic analysis of the dataset in order to identify specific functional classes and co-varying gene sets that might link growth rate and water consumption with adaptations.  In the end, they were able to identify several broad physiological processes likely to contribute to the differences (”nitrogen, proteins and cellwall components biosynthetic and metabolic process as key pathway involved in WUE regulation while sugar,microtubule and phosphatidylinositol biological and metabolic process as key pathway involved in growth regulation. IAA biosyntheticand metabolic process may regulate the drought tolerance”), but I am concerned that the majority, if not all, of these could have been predicted before the study began.

I am well aware of the work that went into this project, and would not like to see it discarded, but I think some changes need to be made to make the data more accessible to naïve readers, and perhaps to clarify the limitations of the experimental design.

The following are some places the authors might consider making changes:

1、I think the first major stumbling block for naïve readers such as myself was the English grammar.  I started correcting it on my copy, but began running into phrases such as one on line 77, “in the middle of the same direction”, that I did not understand, and so could not correct.  Others were more common problems such as the use or absence of “the” (the hardest word in the English language according to the BBC) and plurals singular. I  would strongly encourage the authors to have a language specialist go through the document and correct it to a common standard.  It is possible that if these things didn’t distract me (as a reviewer), I would have been able to follow the arguments more closely;

Answer: I already checked and corrected the manuscript by a professional English editing service. (https://susy.mdpi.com/user/pre_english_article/status.)

I appreciate the efforts the authors took to present their story as clearly as is humanly possible.  I happen to think that the hired editor could have done more to improve the English in this paper, but my views are clearly subjective.  The following are a few things I would have changed if I was writing this (but there are many more examples to point to):

Line 169: I think the word should be processes;

Line 179: I am unsure what was meant by “useful pathway”. This sounds like the authors searched for beneficial or adaptive pathways.  Was this the intent?;

Line 276: Humans “speculate”; GO terms indicate (my teachers criticized me for using “suggest” in cases like this, but the years have passed, I think that battle has been lost.  Nevertheless, even I find speculate too anthropomorphic.);

Line 282: I would insert an “a” before higher:

Line 287: I think resistance should be used here, not resistant;

Line 291: the word should be levels not level;

Lines 388, 399, 400: the phrase should be four types of yellowhorn;

I also would suggest that the title of S1 be changed to “Selected attributes of yellowhorn samples being analyzed”, of Fig S1 to “PCA analysis of transcriptomes of four yellowhorn ecotype”, and of Table S2 to “The number of clean reads mapped to genomes of for types of yellowhorn”.

I believe the word at the lower left of Table S2 should be “multiposition” not “mutiposition”.

2、The second block, that perhaps only affects run of the mill biologists such as myself, was the reference to modules as color blocks (e.g., lines 23 and then 181 onward). I have not seen this before, despite having read and written several transcriptome papers.  I think it deserves some explanation (are these colors standard for specific kinds of networks, or only being used to distinguish the modules in this paper?  If the latter, I think the modules need different names even if some people would argue those names bias the interpretation of the network within them.).  I think it is also appropriate to give the tables in S2 a short legend telling the reader what to learn/conclude from them, and especially, why some text or text background was in red, some in grey, some in green, and many in black and white.

Answer: The modules were divided according to the expression level of DEGs by using R, it is a cluster analysis of the expression level of DEGs. The module colors are random and genes in one module are interrelated at expression level. The networks that we finally obtained were the co-expression network, which is difficult to name according to internal genes. So we modified the relative description in the revised manuscript and explained it as clearly as possible.

Zhang A., et al. Transcriptome co-expression network analysis identifies key genes and regulators of ripening kiwifruit ester biosynthesis. BMC Plant Biology, 2020.

Ahmad S., et al. Morpho-physiological integrators, transcriptome and coexpression network analyses signify the novel molecular signatures associated with axillary bud in chrysanthemum. BMC Plant Biology, 2020.

The added wording helped me considerably.  Thank you.

We already added a short legend in Table S2, and explained the meaning of green background. Other color backgrounds or words only emphasized the genes that appeared in the manuscript, and we deleted them.

That was useful.  I suggest repeating that legend on both pages of the excel file.

3、Several other aspects of the work bothered me, but would be much harder to correct than those above. Nevertheless, if the authors could address any of them, I think the paper could be improved.

Answer: Based on your advices, we modified the manuscript as much as possible.

4、It would be helpful if the authors drew horizontal lines in Table S1 to separate the 4 classes of plants;

Answer: We already added the horizontal lines in Table S1.

Thank you.  it was easier for me to read.

5、It seemed that some of the samples in S1 were meant to be duplicates within each of these classes. Could the authors do a PCA analysis to show how the data sets clustered with each other;

Answer: We already added the results of PCA analysis in Fig. S1, and the trees were divided into four categories according to these indicators.

Thank you. The clustering was amazing given the limitations you faced in how the plants grew.  I think this adds to the credibility of your work.  Very nice.

6、They were working with field-grown plants. Could some of the gene variation seen be due to microenvironmental differences between the plants?  It would have been nice to have sampled the same trees 2 or more times over a given period to see if the expression levels were reproducible.  It might have also been nice to have watered the plants after the drought and then determined which genes were differentially expressed due to the genotype and which to drought (acknowledging there is an important interaction between the two parameters);

Answer: Thank you for your advice. The difference of microenvironment is inevitable in field experiment. In comparative transcriptome analysis, we used five trees of the same type as a repeat, which can largely eliminated these effects. Drought (defense phase) and rehydration after drought (recovery phase) are important and different processes of drought responses, and we mainly concentrates on the former.

I understand the authors desire to distinguish recovery from normal growth.  My suggestion was only to provide the reader with a set of “normal” levels of pathway operation.  I agree no natural group of trees can guarantee that.

7、Faced with 2000+ DEGs, I personally would have increased the stringency to log2³2 or higher so that the discussion could focus on where the biggest differences in DEGs lay. All of these cut-off points are arbitrary.  Once one acknowledges that one can never know all DEGs in any response, or which magnitudes of difference have biological consequences, you can justify why you are only analyzing the most dramatically affected fraction of the DEGs;

Answer: Thank you for your advice. Improving screening criteria, it's really easier for us to focus on more significant DEGs. But It is not conducive for us to obtain genes that are important in drought response but have little change in expression, and construction of co-expression network.

My thought here was to raise stringency criteria so as to reduce the number of DEGs from 2000 to a smaller number of genes consistently showing >4 fold changes in expression.  Some of the pathways would have dropped out but this would highlight those with the greatest differences between the ecotypes.  But I authors’ choices can stand.

8、It would have been useful to confirm the changes that were seen by semiquantitative PCR on a few of the genes using independent RNA collections from the same trees or ones classified in the same way. This would demonstrate the value of this analysis;

Answer: We added the relative expression analysis of six hub genes using RT-PCR in Fig. 7. (Page 10)

Thank you.  Quite meaningful.

9、On line 54, the authors write “Moreover, the study of the physiological characteristics of yellowhorn seedlings revealsthat the content of osmotically regulated substances (soluble sugar, soluble protein and proline) and anti- oxidativeenzymes (SOD, POD and CAT) activity were increased by drought stress [5,14]”. Perhaps the authors could look through their data and determine which specific genes in these responses (as well as those involved in trehalose and sucrose metabolism; mentioned on line 246) are differentially expressed between their different trees, and to what magnitude, and then discuss the meaning of that agreement or disagreement.  I note that the authors did discuss KEGG-defined classes of processes, but I didn’t find the conclusions very clear; I have become disenchanted with the overly generalized groupings shown in Figs 2 and 5. I also don’t benefit from discussions that focus on “DEGs” as if genes showing increases and genes showing decreases in expression can be grouped together.  It would be interesting if the authors addressed specifically which pathways (not processes) were induced, and which suppressed.

Answer: Previous study mainly focus on physiological characteristics of yellowhorn seedlings under drought condition, including the measure of osmotic regulation substances (soluble sugar, soluble protein and proline) and anti-oxidative enzymes (SOD, POD and CAT) activity [5, 14]. It did not contain transcriptome data, and is different from our material and treatment way. Our study focuses on biological pathways and differential genes associated with WUE and growth between different type yellowhorn under a long-term drought condition.

My thought was only whether you see these pathways change at the transcriptome level.  The osmolites you identified are so universal, that any correlation or lack thereof would have been interesting.

In discussion part, we changed the general “nitrogen and sugar metabolism” to specific process, such as “xylan biosynthetic process”. And combined with co-expression network analysis, we further discussed the regulation of these pathways and the relationship of co-expression genes. It is not possible to analyze whether a specific pathway is induced or inhibited based on transcriptome data, but We discussed that genes may positively or negatively regulate a certain pathway to mediate drought resistance. (Page 10-13)

I think this enriched your story.

I also thought the reduction of significant figures in the tables was extremely appropriate.

Comments for author File: Comments.docx

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript has been significantly improved. The Authors have provided missing information or have modified the previous version of the relevant parts of the manuscript. The Discussion section of the manuscript has been enriched with several fragments more accurately describing the transcriptome changes in yellowhorn seedling trees.

Below one slight doubt:In lines 135-136, the Authors wrote, „The qRT-PCR primers are listed in Supplemental Table S3.” But I can not find the sequences of these primers in this Table.”  

Author Response

Below one slight doubt:In lines 135-136, the Authors wrote, „The qRT-PCR primers are listed in Supplemental Table S3.” But I can not find the sequences of these primers in this Table.”

Answer: We already added the sequences of these primers in Table S4.