Studying Gene Expression in Irradiated Barley Cultivars: PM19L-like and CML31-like Expression as Possible Determinants of Radiation Hormesis Effect

Round 1

Reviewer 1 Report

Although this is the re-submitted article, there were still major problems that had not been solved.

1. For practical application, hormesis should be detected in feild experiment. If the authors used etiolated seedlings, the corelation between characteristics detected in etiolated seedlings and plants in feild should have been discussed. However, I could not find it.
2. I had mentioned that summing up scores to make hormesis ranking might not be reasonable approach, but there was no response for that issue.
3. High-dose radiation were not compared in gene expression analysis, thus it was difficult to pinpoint the factors specifically related to "hormesis", not just to broad range of stress responses.
4. Still, I cannot find the result of statistical analysis of expression levels in Table 2. Fold changes higher than 2 were shown in bold, but I could not understand whether these differenses were significant statistically or not. 

Author Response

Dear Reviewer 1,

 In our previous response to the Reviewers, we highlighted all these issues. By some reason that response was not available for you, and we copy our answers to this response, being open for discussion.

 For practical application, hormesis should be detected in feild experiment. If the authors used etiolated seedlings, the corelation between characteristics detected in etiolated seedlings and plants in feild should have been discussed. However, I could not find it.

 In Introduction, we added more information on applicability of hormesis effect in real agriculture. It is not expected to be used in field conditions due to its instability: environmental conditions modify the effect easily. Therefore, we studied “clean” irradiation consequences, without any factors interfering. The usage of etiolated seedlings was dictated by the conditions of transcriptomic research, which was made on etiolated plants. However, our next intention is to study the expression of target genes revealed in this study at all phases of barley ontogeny in the greenhouse.

 I had mentioned that summing up scores to make hormesis ranking might not be reasonable approach, but there was no response for that issue.

To make the data representation clearer, we changed the Figure 1, while Figure 1 from the previous version can now be found as a Supplementary Figure 2. The approach for summing up scores was tested again, considering the real relative change of all traits (Supplementary Table 4) and proved again that the cultivars was divided by three groups – stimulated, almost no effect, and the significantly inhibited cultivar Leon. Inside the group, the position of a cultivar may indeed change, but it does not influence our conclusions, because we discuss the data only in terms of γ-stimulated, γ-inhibited and “no effect” morphological changes. The primary data on all morphological measurements and primary statistics are currently available as a Supplementary Table 2. We also added Section “4.1 Morphological responses of seedlings to seed γ-irradiation” in Discussion and provided strong coefficients of correlation between morphological traits in Supplementary Table 5.

High-dose radiation were not compared in gene expression analysis, thus it was difficult to pinpoint the factors specifically related to "hormesis", not just to broad range of stress responses.

High-dose irradiation consequences are well studied. Current experiment was based on transcriptomic assay of embryos of irradiated Nur cultivar, where we also studied gene expression at high doses of irradiation (doi: 10.1111/jac.12381). According to the goal of this research (low-dose exposure), we used only genes related to low-dose response, excluding those with expression changes, triggered by high-dose.

 Still, I cannot find the result of statistical analysis of expression levels in Table 2. Fold changes higher than 2 were shown in bold, but I could not understand whether these differenses were significant statistically or not. 

Calculations of the fold change of expression (FC) were made using relative quantification by ∆∆Cp method, and fold changes exceeding |2| were considered as significant. This information was added to Material and Methods. Due to usage of three biological replicate per condition, the use of parametric statistics, such as standard error or t-test, would be incorrect. Only minimal and maximal value of expression can be provided in this case.

Reviewer 2 Report

Studying gene expression in irradiated barley cultivars: PM19L-like and CML31-like expression as possible determinants of radiation hormesis effect

Comments and Suggestions for Authors

The updated version includes several corrections; however, it is still necessary to review some of them once more.

You need to include a statistical analysis paragraph in the material and methods section, describing how you implemented t-tests and correlations, as well as the software used for this porpoise.

Specific comments:

Figure 1. It is still difficult to observe differences between groups, breaks on the y-axis could improve the visualization.

STI: Leon cultivar appeared as the least resistant cultivar in figure 2. However, in the description of ST1 it appears that it is very resistant to abiotic stresses, then I am wondering why is behaving so poorly with gamma irradiation.

ST4: Do values on shoot length and root biomass are correct for cultivars eryoma and master?

ST5: There is no paragraph in the material & methods section indicating how you implemented those statistical analyses.

SF1: I recommend translating those labels on the SF1.b

Author Response

Response to the Reviewer 2

You need to include a statistical analysis paragraph in the material and methods section, describing how you implemented t-tests and correlations, as well as the software used for this porpoise.

Information on statistical analysis and software was available in Sections 2.3 and 2.5, now we moved it and created Section 2.6 Data analysis.

Figure 1. It is still difficult to observe differences between groups, breaks on the y-axis could improve the visualization.

Thank you, we added breaks on the y-axis of Figure 1.

STI: Leon cultivar appeared as the least resistant cultivar in figure 2. However, in the description of ST1 it appears that it is very resistant to abiotic stresses, then I am wondering why is behaving so poorly with gamma irradiation.

We do not know the reason and are currently considering this cultivar as a target one for future high-throughput RNA-seq research. So far seems that it can be related to poor ability for double DNA breaks repair. We discussed it in Section 4.2, namely: “Significant induction of SOG1 homologue in roots of the most inhibited cultivar Leon (Table 2) suggests the accumulation of high number of DNA double strand breaks [16] even 7 days after irradiation of seeds, showing that this sensitive to low-dose irradiation cultivar may have less effective DNA repair machinery.”

ST4: Do values on shoot length and root biomass are correct for cultivars eryoma and master?

Yes, everything is correct (please, compare with Supplementary Figure 2, where these cultivars have 121% in comparison with 100% in non-irradiated control).

ST5: There is no paragraph in the material & methods section indicating how you implemented those statistical analyses.

We added this information to Section 2.6

 SF1: I recommend translating those labels on the SF1.b

Thank you, we indeed overlooked this issue. The translation is added.

 Round 2

Reviewer 1 Report

I recommend to accept this paper for publication since authors responded to my comments and improved manuscript. 

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

This paper examined 9 barley cultivars’ growth response to seed γ-irradiation and the expression change of 17 candidate γ-irradiation responsive genes in these cultivars. The goal of this work is to identify gene(s) that lead to positive effect on plant growth resulted from low level γ-irradiation.    

There are major problems in this paper that I think need to be resolved before it can be considered for publication. 

1. The authors didn’t give the reason on why the 9 cultivars were selected for this study. Do they represent a wide genetic diversity in the barley population? Since the goal is to understand the underlying molecular mechanisms and identifying determinant genes, it is necessary to include more samples with great genetic diversity, so the allelic and gene expression diversity could be explored for the goal. It isn’t appropriate to make the statement that “extensive morphological and gene expression analyses were conducted”, based on the fact that they only examined 9 cultivars with unknown genetic diversity and 17 candidate genes, which was identified from a previous study using one genotype, but not QTL study.    
2. When ranking the different cultivars for their irradiation response, the author summed the scores from different measurements. It is very clear from their data that the irradiation response is specific to each measurement and probably also tissue specific. However, by summing up all the scores, such specificity is not taken into consideration and could lead to wrong conclusions. 
3. Then the authors discussed gene expression data based on the summed phenotypic score. I would not consider the conclusion accurate.   
4. While the study collected phenotypic data and gene expression data for both root and shoot, the data was from separate sampling. Although the same irradiation condition was used for both experiments, separate sampling makes it hard to connect the two sets of data. I would strongly suggest generating the two sets of data from the same experiment.  

Reviewer 2 Report

Studying gene expression in irradiated barley cultivars: PM19L-like and CML31-like expression as possible determinants of radiation hormesis effect

Comments and Suggestions for Authors

Dear Editor, in this research the authors analyzed the effect of a hormetic dose of gamma irradiation on seeds of different barley cultivars using a transcriptomic approach. Overall, the manuscript is well written and provides a comprehensive study of the effects of gamma irradiation on the expression of various genes related to defense mechanisms, signaling, photosynthesis, and phytohormone biosynthesis in barley seeds from different cultivars. The research also describes how irradiation specifically affects the stems and roots of the plant once it has germinated. This research is relevant, since it details very well how a hormetic dose can affect different genetic and morphological traits in very different ways on the same plant species. Therefore, it can significantly affect the development of agricultural treatments in which this type of irradiation is applied. Thus, it could be suitable for publication in Agronomy after minor points.

General comments:

Even if it is stated in some sentences, I think that a small section is necessary to compare the gene expression among shoots and roots, within cultivars in the different categories mentioned.

Do you have any picture comparing the roots and shoots of the Leon and fox1 cultivars? I could be a good supplement.

Specific comments:

Line 16: Include Gamma (γ)- irradiation

Line 24: reconsider the sentence as follows: showed that the genetic background of a cultivar plays an important role in eustress

Line 29: Consider “Induce” instead of “provoke”

Line 31: Consider “beneficial” instead of “positive”

Line 32: change “low” for “at”

Line 35: Reconsider reordering: In agriculture, exposing plants with stimulating doses of γ-irradiation is a promising approach

Lines 38-40. Consider reordering:  The potential of a given radiation dose to elicit eustres or distress responses depends on many factors, including the plant species, cultivar, stage of development, radiation quality, as well as many others.

Line 45: Consider the following sentence: It has been observed that exposed seeds to … do not use the term rule when only citing a single study

Line 47: “at” instead of “in”. Please check the prior in the rest of the text.

Line 48: check punctuation: damage allows mobilization of endosperm resources, even under limited growth conditions

Line 50: Missing preposition. irradiation of barley seeds in the dose range of 15-20 Gy induced

Line 52: Consider the following changes. shift on, as well as

Line 55: Consider wording. Therefore, to elucidate the molecular

Line 66: at what temperature and RH were the seeds irradiated?

Line 68: consider, “treatment”

Line 71: the development of the shoots was also in the dark? Was a growth chamber with light required?

Line 96: please check how to quote a software

Line 120: Use sentence case format in the full name of SOG1

Line 124: idem line 96

Line 126: please check syntax. µL instead of µl

Line 140. Figure 1. axis titles and legends should be larger, the scale on the y-axis should include 0 and then a break to appreciate the differences. Why does the frame have different colors? If it has a purpose, it should be included in the legend.

Line 145: missing Article The figure 2

Line 148. It is difficult to follow this type of plot, probably a multiple bar chart is more convenient.

Line 160&161:  Fig. 2 instead of Figure 2

Line 166: please check the sentence: The gene expression analysis included ……….

Line 170: seven instead of 7

Line 172 please consider reordering the title: changes in gene expression (folds) in shoots and roots of barley seedlings……..

Line 172: for a better visualization of the table, delimiters could be included among the classes

Line 178: This sentence should be placed in the discussion section

Line 199: This statement is not clear since there are not values in the table. Why there was no expression detected?

Line 219: Absicic acid (ABA) signaling, citation missing, and this sentence is probably better placed on the discussion section

Lines 263-264: Citation missing

Lines 268-269: Only due to that repair mechanism?

Lines 314-318 These adjustments to regulate the redox metabolism apply only for the Eryoma cultivar? The changes on gene expression and the ROS synthesis are not well explained in this paragraph, starting from line 303.

Line 330: What does high plasticity means? High quality seeds were also used to obtain roots and shoots from the rest of cultivars

Line 346: PM19L-like and CML31-like genes were considered.

Line 347: cultivars

Lines 358-360: please rearrange this sentence

Lines 398-400: To search for possible ….we’ve studied…..

Comments for author File: Comments.docx

Reviewer 3 Report

The authors studied the mechanism of hormesis which has not been studied well despit of its high portential for application in agronomy. However, I think there were several important problems in design of research and description of results as follows;

1. The analyses were performed only for etiolated seedlings. The growth condition was very different from normal growth condition under which hormesis may be applicable in agriculture. It is difficult to agree that the plants with longer or heavier shoot in dark condition always also show vigor in light condition. For the practical significance, the growth rate in normal condition should be also presented to show the relationship with characteristics detected in dark condition.
2. Four traits analyzed in this study were not directly related to each other, thus, it seems not to be correct to rank cultivars according to sum of score for each trait. Many cultivars showed totally different responses according to traits. At this point, it was difficult to get clear definition of "hormesis" in this research.
3. The groups irradiated with high-dose radiation were not compared in gene expression analysis. Therefore, it was difficult to pinpoint the factors specifically related to "hormesis", not just to broad range of stress responses.
4. In table 2, result of statistical analysis was not provided. The difference of expressions, which is the most important factor in this research, should be shown to be significant by statistical analysis.
5. (minor issue) A figure showing classification and expression level of target genes may help readers to understand the result and evaluate validity of conclusion at a glance. In present form, it is very difficult to link the role of each gene and thier expression pattern.