PONE-D-23-33008The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryosPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled “The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos” examined the impact of abiotic stress conditions either alone or in various combinations on ns-LTP2.8 gene expression level and its protein synthesis in vegetative tissues and embryonic axis of selected barley genotypes. However, there are few concerns which need to be addressed by authors before it can be considered for publication:

Comment 1 : Authors should conclude their study highlighting the major outcomes, significance as well as future prospects of the study . A separate conclusion section should be added.

Comment 2: As authors have performed real time PCR for expression analysis, The term RT-PCR used in the manuscript should be replaced with more appropriate term RT-qPCR.

Comment 3: Authors should mention in the manuscript why they have selected only one concentration of PEG and NaCl for application of drought and salinity stress? Have they screened for response to other concentrations?

Comment 4: Authors should mention in the figure legends of Figure 5, 6 and 7 which gene was used as housekeeping/ endogenous control for data normalization and how many replicates were used.

Comment 5: Legend of Fig 2 should be re-written as: Agarose gel photograph showing PCR amplification of ns-LTP2.8 gene from different barley genotypes. For each genotype, three replicates were used for PCR reaction. Red marks indicate saturated pixels in ChemiDoc™ XRS+ System (Bio-Rad). M represents 100 bp DNA ladder.

Reviewer #2: - The design of the experiment is relatively good. A lot of work has been done.

Comments:

- The manuscript needs English polishing

- The manuscript is very long and tedious for the reader. It is necessary to remove unnecessary parts and clean the remaining parts.

- The abstract of the manuscript can be written better. For example, authors should not provide an interpretation or conclusion about the gene before they describe their work. Also, the abstract is currently not a good representative of the study and does not have the impact it should have on the readers to encourage them to read the manuscript.

- The introduction is fragmented and suddenly jumps from one subject to another—for example, lines 31 to 34. Also, nsLTPs in plants and their role in plant development and response to environmental conditions have yet to be discussed. Most of the focus is on their allergenicity.

- Plant materials: good explanations are given. Is it possible for the authors to summarize the information in a table? Also, is there any information about the response to stresses for the first set of plants?

- How was the salt solution added to the pots? All at once or gradually? From above or below?

- How are the root samples separated from the soil?

- Which leaves were used for sampling?

- Figure 1 does not help to provide information.

- Figure 2 should be presented in the results. Also, please add negative and positive samples to the figure.

- Line 210-211 is repetitive. They were given in the previous section.

- Examination of gene expression: has DNA contamination been removed from RNA samples?

- Please provide the ANOVA results in a supplementary table. It is also better to compare the means with one of the more well-known tests (Duncan, LSD, etc.). If the interaction effect of the treatments is significant, the comparison of the means of the individual effects is not statistically correct.

I suggest additional statistical analyses (correlation, PCA, etc.). These analyses can show the relationship between the measured traits and the stress response. For example, has a relationship been between stress tolerance and gene expression or protein amount?

- About qPCR internal control genes: EF1a gene amplicon is longer than usual, and UB1 gene amplicon is shorter than usual. Please justify this.

- Figures related to gene expression analysis could be clearer. It needs to be clarified what the control is.

- As expected, the results have shown that the gene is not expressed in the vegetative tissues of the plant. I suggest the authors analyze this gene's promoter so that they can provide possible reasons for this lack of expression.

- But my most important question to the authors:

How can they prove with the presented data that the change in expression and amount of protein is effective in stress tolerance?

Reviewer #3: Summary: The data presented in this manuscript are part of a two important scientific problems: (1) abiotic stresses have major impacts on plant productivity and understanding how plants respond to stress is necessary to develop future crops in a warming world, and (2) accumulation of compounds for stress mediation may have unexpected consequences, i.e. increase in human allergens in stress-tolerant crops. While these are important problems to study, the scope of this manuscript is narrow – they measured transcript and protein levels for a single gene and gene product in 12 barley genotypes in response to eight stress conditions. While it is fairly narrow, this does represent a lot of work on behalf of the authors and is likely to be important for future research. I have a few recommendations for increasing the clarity and presentation of the data to improve the manuscript.

About barplots: in figures 5, 6, 7, 8, 9 and 10, and supplemental figure S3, results for multiple data points are presented in barplots with error bars. The authors should instead use a method to show the distinct data points, e.g. univariate scatter plots. For reasoning why I make this suggestion, please read “Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm” from Weissgerber et al 2015 (PLOS Biology 13(4): e100228, doi: 10.1371/journal.pbio.1002128).

Abstract: please better describe nsLTP-2.8, i.e. what it is and why it was chosen. The story that it is a food allergen that accumulates in grains and is a lipid transfer protein that plays a role in abiotic stress response is compelling. But as a reader, I did not understand these things until I read the introduction. This information should be made obvious in the abstract to better sell why this work is important.

Line 218: sequence data for RT-PCR product “data not shown”. Please show this in supplemental data.

Please provide an explanation or evidence that anti-nsLTP-2.8 antibodies do not bind other nsLTP-2 proteins. Particularly, the region in nsLTP-2.7 looks very similar – 11 out of 16 residues are identical, 3 out of 16 are strongly similar (residues 2, 4, and 5), and only 2 out of 16 (residues 14, 15) have no identity. Would the size of nsLTP-2.7 be notably different on the western blot gel?

Figure S3 represents the nsLTP-2.8 transcript level in all genotypes, aggregated into a single panel. These data should instead be shown individually – one panel for each genotype.

Reviewer #4: The paper was mainly about the influence of various abiotic stresses acting separately as well as in their combinations on the change in the ns-LTP2.8 transcript and ns-LTP2.8 protein contentsin the embryonal axes of germinating spring barley genotypes with different ns-LTP2.8 allelic forms and stress tolerance. I found it so much impressive and to innovative. However, I have some comments as below:

Abstract was soundly fine. The introduction chapter was organized and well justified.

l. 49-51, 66-67 requires citations; l. 71 authors are advised to spell out ‘GPI’;

Method section was well described, especially when they properly elaborated the approach on that basis they combined different stress factors (l.148-151). They have mentioned that stress endeded when radicle reached to the length of 2 mm. I suggest them add a citation here (l.152). In l.159 the expression "IPG PAS" possibly stand for a location or a study filed name: I recommend authors to clarify here. l. 167. a short description of BBCH sounds necessary here for readers. A spelling out for BSA and TBS terms in l. 247 and l.264, respectively is suggested. l. 336. the mentioned result requires evidence (by adding a Fig, number). Discussion chapter was properly explained their results with reasonable descriptions which was nicely associated with their findings and other results from earlier studies. Their suggestion for future studies was also impressive (l. 497-499). It can also be said that their combined stress factors could be considered as the innovative part of the study as it is most likely the case in natural conditions. In l. 549. the sentence edning with "ns-LTP2.8 gene was observed" is missing a full stop. A short conclusion for what was reported here seems necessary. Also, as my last suggestion, it could be much more interesting if they could explain a bit more about the relation between high temperature and drought stress factors in connection with salinity stress when they were explaining the opposite relationship ( not mandatory ).

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Peiman Zandi

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The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos

PONE-D-23-33008R1

Dear Dr. Kuczyńska,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Shailender Kumar Verma, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have addressed all the concerns raised previously. However, authors need to take English language editing services as there are many grammatical errors in the entire manuscript. All those need to be corrected before manuscript can be accepted for publication.

Reviewer #2: (No Response)

Reviewer #3: Authors have adequately addressed the issues I previously raised. I appreciate the inclusion of the technical screening figure showing the seedling shoot western blot results, and I recommend that the reviewers include this image as a supplemental figure with a short description in the manuscript text to ensure readers have confidence in the authors’ antibodies. I also recommend providing the information about the differences of nsLTP-2.7 and nsLTP-2.8 molecular weight and localization in the manuscript text.

Reviewer #4: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Peiman Zandi

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