Biochemical and Gene Expression Analyses in Different Poplar Clones: The Selection Tools for Afforestation of Halomorphic Environments

Round 1

Reviewer 1 Report

Galovic and co-authors present biochemical and gene expression analyses to four poplars subjected to different salt stress conditions. Given that these poplars could be used for afforestation in salt rich soils, the authors try to understand which could be possible good candidate clones based on their stress responses. The issue is pertinent and interesting. This new version of this article would still greatly benefit from re-writing and re-styling. Reducing the extension of its introduction and results sections would also benefit the work, that reads too much as preliminary. Little changes were made from the last version. Some concerns in relation to the following aspects:

1. Was only b-actin reference gene used? There should be at least 2 reference genes according to MIQE guidelines; maybe actin was not a good reference gene to use for clone Bora, and this would explain the downregulation of all genes in the Bora clone. Or perhaps the quality of the RNA/sample in this clone was not good enough?
2. The choice of your statistical analysis to the data. These post-hoc tests are invalid for this type of data. This type of analysis does not make sense and makes the data not interpretable. This type of data requires different type of analysis, such as GLM or GAM/GAMM (particularly evident in the difficulty to follow Figure 1-4 statistical data). There are too many factors and variables to be analyzed by these statistical tests. I suggest finding a more adequate and clearer way to analyse these results. Also Figure 8 data needs some statistical analysis.
3. Line 526-533 This description should be included in material and methods and/or caption to the corresponding figure.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The aim of the reviewed work was to evaluate the biochemical and molecular methods that can be used in phytoremediation of halomorphic soils. The authors showed that the genetic variation of the Populus clones was reflected in their response to salinity stress. Since 2006, when the genes of the black poplar (Populus trichocarpa Torr. & Gray) were sequenced, it has been possible to identify candidate genes that underlie the physiological processes responsible for the organism's response to environmental stresses. In this article, such genome-based studies provided insight into the genetic mechanisms of adaptation to salt stress. The results presented in the study are interesting and may help in the remediation of saline soils not only in Serbia. From this point of view, this is an important article, but requires careful preparation of the discussion and removal of numerous errors, both editorial and linguistic. All figure captions require correction and the charts themselves should have a more readable form. The letters they contain are too small and illegible. The methodology applied is correctly described and the results are of some interest. All detailed comments are included in the attached pdf version.

Comments for author File: Comments.pdf

Author Response

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Round 2

Reviewer 1 Report

Dear Authors,

Thank you for your reply. As mentioned in my previous review, Figs. 1 – 4 are not interpretable. One of the biostatistics basic rules is parsimony. You have too many factors to conduct post-hoc multiple-comparison tests when you should be analysing the effects of variables and the contribution of factors to explain overall variance. For such reasons, you are better advised, e.g., to perform analysis such as GLM followed by ANOVA-GLM based on deviances. However, I call for an editorial judgement on these figures to see if it is clear and reasonable to have such large sets of letters on top of bars. By the way, please let me bring to your attention that Tukey’s is not an “adjustment” but rather a multiple comparison method, which is not really what you need here. Alternatively, focus on the results from the 3-way ANOVA you conducted, put them in a table (include dfs, statistics, p-values, etc for each variable) and draw your main conclusion from there. Then break your plots into multiple panels instead of meaninglessly comparing everything with everything. I think insisting on such Figures is not fair to the work.

Author Response

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Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The topic covered by the manuscript is very relevant to current concerns regarding climate change and is likely to attract future attention. The introduction covers the topic broadly and clearly introduces the different candidates appropriately. My main concerns are the materials an methods section. The experimental design would need to be described as well as a better description of sampling, replicates, condition of the samples, etc.

My main general concerns are:
- The manuscript seems to offer as a sole solution the genetic modification of poplars. As many countries are not very open to allow transgenic plant material, especially in forests and natural ecosystems, I suggest that the authors mention the possibility of breeding and further selection of candidate genes in the offspring. This is a more broadly accepted approach.
- I suggest that only the clone code or name are used throughout the manuscript to avoid confusion. Having a double nomenclature is confusing. The names can be stated in the materials section of the authors wish so, for the rest of the manuscript I recommend the clone codes.
- Materials and methods section needs to be improved. Vague terms like certain and various are used instead of numbers. Additionally, the sampling of plant material and experimental design needs to be described.
- There seems to be something going on with clone Bora in the expression study. all genes seem to be down-regulated in all cases. This, in my opinion, indicates a failure of the reference gene.
- General note, gene names should be reformatted to italics throughout the text.
- General note, Species names should be italicised throughout the text.

Minor comments

Line 19, Poplar,correct
Line 30, Are the five candidate genes presented afterwards. This way it seems that five candidate genes and tow, one and two genes were tested, a total of 10. Please, correct for clarity.
Line 38, will be seems to immediate. Even though they are strong candidates, a transformation experiment would have to be carried out to validate these candidates before developing salt-tolerant clones. I suggest to change the statement with something milder like “Are promising candidates for salt tolerance in poplar.” or “are good candidates for validation through generation of transgenic poplar lines.”
Lines 42-43, This seems to be a very strong statement. Perhaps “directly puts under distress”. In any case this statement would need examples of important forest species which are on the brink of extinction or threatened.
Line 72, What number? If there are three it is better to present it as three. This will help the reader expect what comes next.
Line 88, molecular-based, correct
Line 89, that has drawn, correct. if only one study has been carried out, “a lot” is an overstatement. I would suggest “that has drawn scientific attention”
Line 100, amino, correct
Line 142, if the authors are talking about SOS1, indicate it at the beginning of the sentence.
Lines 283-285. Gene expression analyses: Were samples extracted fresh or were they frozen in liquid nitrogen and ground prior to RNA extraction? More information regarding the sampling of the leaves is needed in this section. How many technical/biological replicates? How many treatments? How many time points?
Line 322, How many house keeping genes? What are their names? “Various” is not acceptable when describing a methods section. Was only B-actin used as described below?
Lines 323-325, The use of certain in this section needs to be avoided. Numbers are needed. certain is a vague term not suitable for scientific writing.
Figures 1-6. These figures should include standard deviation. The legends of the columns that illustrate statistical categories are not indicated in the figure legends. Not sure a comparison of everything with so many statistical categories is useful to the reader or to present the results. Remove horizontal grey bars from figures.
Line 467, tolerant. Correct
Line 445, 478 and 480, peak. Correct
Figure 7.S Should this figure show the Arabidopsis proteins as well, as they are the ones for which a function has been validated?
Lines 526-533. This information should be split between the methods section and the legend of Figure 7. The results and discussion section should not include these details.
Figure 8. I recommend splitting the Y axis in figures for which all values except for one have similar values, as it is difficult to see differences between treatments. PtGRAS17 is missing in Bora.
Figure 9. Why are there several 150mM, 300 mM and 450 mM but only one control? If this reflects time points control should also be done at different time points, if they reflect technical replicates I recommend merging replicates.
Line 537 and 575 both are point 3.2.1, correct.
3.2.4. Was the efficiency of the primers in the two homologues compared? This could affect the results. Are these homologs represented in Fig 7?

Reviewer 2 Report

 

Galovic and co-authors present an abiotic stress study by performing several biochemical and gene expression analysis to four poplars, that are clones of interest for afforestation in salt rich soils. The science in the manuscript is hard to evaluate, mainly because this manuscript reads too much as a school report or a thesis, very descriptive, no storytelling. The article would greatly benefit from some re-writing and reducing the extension of its introduction, mat&met and results. There are several redundant sentences, some editing and critical review is advisable. As it is, it is very difficult to evaluate the science, it is very preliminary at this stage and should be re-written to be in a publishable scientific writing format. Below, I give some examples where improvement must be done, but overall editing and re-writing of the manuscript is needed throughout before being evaluated for its scientific soundness.

For instance, from line 129-132: The following two sentences are not needed at this point, it could be reformulated.

The Mat & Methods section needs revision. Could the authors alter the order of appearance of the gene studies related sections in Mat&Met. The first section should be the selection of genes, maybe start by the description of phylogenetic analysis in 2.5.1 (that appears in Results section but has no corresponding description of the analysis in Mat&Met). Followed by section 2.4.1 and 2.4.2. Gene expression data analyses, that needs to be referenced and relative expression method used for calculations clarified. Also, text would greatly benefit from some editing work, reducing redundant sentences.

 

Take care to verify each gene ID given on table 2, for instance XM_034932549 is not beta-actin.

 

Line 341-349, do not seem to belong to the end of material and methods, it reads as introduction material.

 

In the 3. Results and discussion section and subsections, the authors should make an effort to divide the sub-sections of results in terms of the main result obtained. The manuscript reads too much as a school report, repetitive, boring and I advise the authors to re-writte the section titles and part of the sections themselves.

Each section should start off thinking on the following terms and telling your story: What is my scientific question? To answer this we did this experiment. Description of results and a conclusion. Does it raise new questions?

For instance, instead of 3.1. Biochemical analyses, maybe subdivide the sections according to the main conclusion of each section, what is your main result for this or that experiment?

This section should start in Line 367, and Figure calling embedded in the text, not as it is, where the Figures are described in a long first paragraph in the section.

 

 

Minor comments, some typos detected.

Line 19: please correct where it says “Serbian polar clones…”

Line 21: please correct where it says “Three perspective clones  “

Line 100: please correct: where it says “immino acid”

Reviewer 3 Report

In my opinion the results and discussion is not presented clearly.

Also, a native English speaker should have a look on the manuscript. Sometimes the sentence structure or other issues don't allow to understand the meaning of a sentence or the motivation for a statement.

I would like to see more details about how the qPCR step was performed. Quantity of RNA used in reverse transcription is not indicated. RNA integrity is not indicated. Quantity of cDNA (or dilution factor of reverse transcription sample) not mentioned. Amount of technical replicates in qPCR (and number of samples tested for each treatment and time point) not mentioned. Only one endogenous control gene. This is typically considered insufficient for a publication. Maybe you have data like Ct values for the beta actin gene? If they would be clearly uniform for all the samples, that could be used to support the quality of the experiment.

Figures 1-6 would benefit from redesigning, maybe, by introduction on a y axis on the right side which would represent values in % change from control (which you so often use in the results and discussion section).

What is meant by the small letters on the bars in Figures 1-6 (by the way not all the bars have them).

I don't think there is much use of Figure 9 at least in present quality.

I have several more notes and comments which I attached in the scanned file.

I don't think that this manuscript is fit for publishing in the current form.

Comments for author File: Comments.pdf