Exploring Reactive Oxygen Species Accumulation in Allium fistulosum L. Seeds Exposed to Different Storage Conditions
, Anca Macovei 2, Adriano Ravasio 3, Andrea Pagano 2, Conrado Jr Dueñas 2, Xianzong Xia 1, Roman Hołubowicz 1 and Alma Balestrazzi 2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsResearch topics of manuscript is interesting. ROS (reactive oxygen species) accumulation in stored seed is among the main causes of deleterious changes observed in aged seeds. The increase in ROS level in Welsh onion seeds may explain why these seeds lose viability during storage.
In this work, the ROS level was studied in seeds of 14 genotypes of Welsh onion. Seeds were stored under 6 storage conditions, differing in RH (air relative humidity) and temperature, for 12 and 22 months. Then the ROS level in stored seeds was determined. However, there are no information on the ROS level in seeds at 0 months of seed storage (control seeds). Without knowing the initial values, it is difficult to assess whether the ROS level observed after 12 months was a consequence of storage or whether it resulted from the diversity of seed genotypes (initial ROS content in seeds) used in the study.
There are confusing sentences: in the abstract “Seed samples were stored for 30 months under six different conditions of temperature and relative humidity” (line 17), and in the Materials and Methods “Seed samples were stored for 30 months under six different conditions, as shown in Table 2” (lines95-96), whereas the results of work indicate that the seeds were stored for 12 and 22 months.
It is unclear what parameter is presented in the charts 1 and 2. What does the term “humidity” mean? Is it the seed water content? How was this parameter expressed (units)and how was its value measured?
What values are presented on the charts 1 and 2, whether they are the means? Why are the values of SEM or SD not presented?
“It is worth noting that the ROS levels detected at 22 months were significantly higher compared to those detected at 12 months of storage whereas the estimated humidity values were decreased” (lines 180-182) – what statistical test was used to confirm statistical significance differences between the levels of ROS in seed stored for 12 and 22 months?
Whether and how was the statistical significance of differences between ROS level in seeds stored at different conditions investigated? If and what statistical test was used? – The results of such tests may indicate the conditions under which the ROS level in the seeds was the lowest and which are potentially most favourable for the storage of Welsh onion seeds.
Whether and how was the statistical significance of differences between ROS level in seeds of different genotypes/varieties investigated? If and what statistical test was used?
“Long-term storage is associated with increased lipid peroxidation, reduced levels of antioxidants. Ideally, Welsh onion seeds can be stored for 5 to 15 years if maintained at 2°C or for 15 to 50 years when conserved at -18°C” (lines 235-238) – lack citation.
“The ROS profiles produced by the Welsh onion seeds can provide some interesting information about the cellular events associated with imbibition, possibly related to different storage conditions and genetic background” (lines 242-244) – this sentence is unclear, was the level of ROS tested in imbibed seeds in this work?
Comments on the Quality of English LanguageMinor editing of English language ris equired.
Author Response
See attached file with the detailed response to reviewer
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript by Padula et al. focuses on the short-lived species Allium fistulosum and has the merit to bring to attention two relevant aspects for Seed Biology. The first is the non-destructive detection of ROS, and the second covers the characterisation of seed longevity under a set of six storage conditions used by the seed trade. The experimental design was quite elaborate and included 14 seed lots differing for production and breeding sites and genetic background, to account for parental influences on seed quality.
Nonetheless, it remains an exploratory study (as declared in the title), which requires some considerable revisions prior my recommendation for publication.
My comments on the various manuscript sections are listed as follows.
Abstract
To improve structure and readability here, I suggest starting the abstract by combing the two sentences from l. 12 to 15. The purpose of the study should follow and lines 11-12 could be condensed with line 17-18. Moreover, it is relevant to specify the range of storage conditions tested (temperature and RH).
Importantly, the storage interval of 30 months is not consistent with data shown in the results and should be well clarified. Finally, the values (or range) of temperature and RH should be declared in the final statement.
Introduction
1. Welsh should be capitalised throughout the manuscript.
2. L. 38-39 Remove “..and the added value of A. fistulosum cultivation” to make the sentence clearer.
3. L. 41. Did priming improved germination and vigour compared to fresh seeds or after storage?
4. L. 42 Gap of knowledge in what?
5. L. 43 Rather than seed viability under storage, I recommend using seed storability/longevity/lifespan.
6. L. 47 Remove space after “from”.
7. L. 49 Change to “during the early phase of germination, which otherwise will fail” and provide reference on seed DNA repair.
8. L. 51-62 I find this discussion interesting but risky, as it may raise expectations on DNA and chromosome analyses, which are not part of this study. I suggest shortening and simplifying.
9. L. 70 Conclude by adding “conditions of temperature and RH relevant to the seed horticultural trade”.
Materials and Methods.
10. L. 89 7-8% on a fresh weight basis? Please, clarify.
11. L. 90-91 Had all genotypes/seed lots germinated more than 90%?
12. L. 94 “Covered” – use past tense. The temperature and RH ranges are confusing and do not match with the values of Table 2, showed in the results, and discussed.
13. L. 99 Photoperiod instead of phases.
14. L. 130 Were the assumptions to run Pearson’s correlations checked and fulfilled?
15. L. 135-137 Unnecessary and not part of statistical analysis. Remove.
Results
16. L. 142 Which germination data were considered (first or second count)? Was there any significant difference between the two counts?
17. L. 143 Remove “It is possible to observe how” to reduce wordiness.
18. L. 144 Higher than what? It does not seem the case for storage condition A.
19. L. 144-150 These statements are not statistically supported.
20. L. 157 Remove “to analyse the relation”.
21. L. 159 Use revealed instead of evidenced.
22. L. 160 “The majority” (article missing) and “were” instead of are.
23. L. 171 Storage interval instead of timepoint (here and hereafter) and delete “the observed”
24. L. 172 Enhanced.
25. L. 171-177 Statistical support is missing. No italics in for temperature values.
26. L. 174-178 It reads unclear compared to what ROS levels were enhanced.
27. L. 179-180 replace with “by all tested storage conditions”.
28. L. 180-182 Statistical evidence required.
29. L. 182 Remove “were”.
30. L. 207 It is more precise to refer to total germination rather than germination % here and in the rest of the manuscript.
31. L. 207 “was” and not “is”.
32. L. 186 Seed response is too general. Use seed viability or germinability.
33. L. 188-189 Remove “as evidenced in the graphics”.
34. L. 204 Change to “To provide an overall data interpretation”
35. L. 205 Remove plot and use “were” and not was.
36. L. 208-210. Abbreviations in parentheses are confusing. Please, remove.
37. L. 208 “after 22 months” instead of “at the second sampling time”.
38. L. 210-211 Replace with “This may indicate that”, and use the past tense (were).
39. L. 214 Remove “taken into consideration”.
40. L. 215-216 Move “of the total variance” after 71.2%.
41. L. 217 Interestingly inst4ead of “it is interesting to note that…”
42. L. 218 the two storage intervals instead of “sapling I and II”.
43. L. 219 were much more divergent.
44. L. 221 Use tested conditions and remove “taken under consideration”.
Discussion
L. 231 Germination performance was not assessed in this study, as it would at least require measurement of speed of germination. Therefore, authors can only talk about germination and/or seed viability. The same applies to L. 263.
L. 233 Comma missing before “makes”.
L. 234 Remove under a range of conditions.
L. 236 Something is missing in the sentence, including supporting references.
L. 238 References supporting the statement should be provided.
L. 242-244 I could not understand this sentence. Was it referring to primed seeds? Please, revise.
L. 246 Full stop in missing.
L. 247 Specify “ROS accumulation in dry seeds”.
L. 248 Viability or germinability instead of response.
L. 251-254 The structure of the sentence is not flowing, and authors should also cite topical reviews/ book chapters on redox biology (ROS, RNS).
L. 258 Missing reference on original studies/ book chapters on the Fenton reaction.
L. 260-261 That ROS accumulation was attenuated under storage conditions D and E remained unclear to me. Compared to what? Was this indication statistically significant? Which kind of analyses would then be conducted under such conditions?
Conclusions
L. 277-279 I am not sure that the storage protocols (if under controlled conditions) would help overcome seed lot and genotype variability, as this are more related to the initial viability (as per seed viability equation). I am not sure to have caught the right message here and authors should clarify on this.
References
There were some typos and formatting mistakes (eg, sometimes species were not italicised), which need to be fixed. Importantly, 30% of the reference are self-citations. Although, they were appropriate, authors should expand their references list acknowledging more the works by other colleagues on similar topics (eg, ROS, seed longevity).
Figures and captions
Table 1. Specify in the caption what F1 stands for E and F.
Table 2. Exact values of temperatures are needed. Were not seed water contents measured? Why?
Figures 1 and 2. Error bars are missing for all variables alongside statistical information. It is conceptually wrong to connect categorical variables with lines (ie ROS and Humid) and data representations should be revised. It was unclear if the germination values refer to the second or first count and if differences were detected. The font was in general too small, and I suggest labelling the y-axes to avoid confusion. I am not sure what Humid (please replace by RH through the figure – it is a more standard and immediate abbreviation) referred to and how it was measured. Did authors report fluctuations of RH inside the incubator chambers?
There is no need to reiterate identical legends. One bigger legend on each figure is enough. I also suggest indicating the T and RH values on top of each panel, instead of referring to Table 2.
In the caption, authors should refer to total germination.
Tables 3 and 4. Capitalise or not P-values according to Seed guidelines. Use Person's r and replace evidenced with “shown”. No red was visible on significant values. Perhaps bold or asterisks are betters option. Use RH instead of Humid in all Figures and captions.
Figure 3. The font of the legend in panel b was too small, and authors should refer to Table 2 in the caption to explain the seed lot abbreviations. In the caption remove “each measured parameter”. Specify to what P-values are referring (I assume r coefficients).
I am confident that authors can address all my remarks and hope they find them useful to add value to their work and improve the quality of their manuscript.
Yours sincerely
Comments on the Quality of English LanguageSome typos, punctuation and minor grammar mistakes. The language was sometimes unnecessarily wordy, and some suggestions to improve conciseness were provided in the comments.
Author Response
see attached file
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors1. The MS entitled, Exploring Reactive Oxygen Species Accumulation in Allium fistulosum L. Seeds Exposed to Different Storage Conditions deals with the optimization of storage conditions of Allium fistulosum L. Seeds based on accumulation of ROS. Authors have subjected selected seeds to combination of temperature and humidity conditions such as- A 175 (25°C, 25% RH), B (25°C, 45% RH), C (10°C, 25% RH), E (< 10°C, 25% RH) and F (< 10°C, 176 45% RH) and measured ROS level. Authors have studied the response of Welsh onion seeds of various origin, subjected to different storage conditions, by combining germination performance with the seed ability to accumulate ROS. Kindly address the following suggestions to improve the MS.
2. Abstract- Add different conditions used for optimization.
3. In Introduction, mention if any other researcher has used ROS level of seeds for optimization of storage conditions.
4. In Materials and methods section, description of principle may be reduced or omitted.
5. In Figure 1 legends mention full forms of Germ & Humid.
6. I am not convinced with the data representation. I feel instead of comparing parameters, authors must compare different storage conditions and conclude which storage condition is best. In addition, the result can be confirmed with seed viability test.
7. Discussion must be focused on how estimation of ROS is beneficial as compared to other techniques used for optimization of seed storage.
8. Add a picture of seeds, seed germination test.
9. References must be uniform.
Comments on the Quality of English Language
Critical proof reading is required.
Author Response
see attached file
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThis work investigated the production of ROS in A. fistulosum seeds stored under six different conditions of temperature and relative humidity, and show that ROS accumulation is dependent on storage condition. But the manuscript needs improvement:
1. The authors did not conduct in-depth analysis and explanation of the experimental results, such as in line 160 “Majority of observed significant correlations are between ROS and germination, and not related to humidity.” How is the correlation between these data and germination? Clear description and summary should be provided. There are many similar situations in the results that should be modified.
2. The discussion section of the manuscript is insufficientï¼› it needs to be more fully compared with relevant researches. The conclusion section also fails to provide a clear statement on the optimal seed storage conditions and suitable commercial application suggestions.
3. In the caption of Table 3 and 4, significant differences indicated ”evidenced in red” but is not displayed. It is recommended to use asterisks or other markings instead.
4. There are spelling and punctuation errors in the text that need to be checked and corrected.
Author Response
see attached file
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease see the attached file.
Comments for author File: Comments.pdf
Author Response
The manuscript has been revised, chan ges are highlighted in red
we hope we have fully addressed the Reviewer's requirements
best regards
Alma Balestrazzi
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsI acknowledge that the authors improved their manuscript by shortening and fixing most of the stylistic/terminological suggestions. Nonetheless, I believe the manuscript retains major points that require to be addressed prior my advice for publication.
My comments on the various manuscript sections follow here below.
Abstract
L. 13-15 The added sentence still shows a structure not very fluent. Furthermore, I would use “can” rather than “will” in line 15.
L. 18-19 I believe that referring to the various storage conditions using letters do not help readers in the context of the abstract, whereby they are not later used.
L. 20 After 12 and 22 months of storage rather than “at”.
L. 22 Increased ROS levels instead of “enhanced”.
L. 24- 25 “those temperature and relative humidity values with a lower impact on seed ageing”. Once more, I recommend the author to indicate those values. Despite the purely explorative nature of their study, authors should provide some findings to the readership, who would reasonably expect more details here.
Introduction
1. L. 49 Remove under storage. Seed longevity is defined as maintenance of viability under storage.
2. L. 55 Germination of (not in) Welsh onion.
3. L. 55-59 I think it would be useful to clearing report the storage conditions used in references 13, 14, and 15.
4. L. 58 Use Greek letter for “gamma”.
Materials and Methods.
5. L. 87 Seed moisture content rather than “moisture level”.
6. L. 92 “spanned” instead of “covers”.
7. L. 93 Stored for 30 months. I am still confused, as the storage intervals used to measure ROS levels and germination were 12 and 22 months. What happened to seed left after 22 months up to 30? Is this a slip left in the text?
8. L. 185-187 My previous comment was meant to say that this text is unnecessary and not related to any statistical analyses. Therefore, I would recommend removing it, also considering that the type of representation reproposed is conceptually wrong for ROS and RH values.
Results
Prior to discussing any germination data, the authors should declare (and possibly show, perhaps in supplementary material) that they found no statistically significant differences between the first and the second count. They should also mention in the manuscript that data shown in the main figures refer to the second count, similar to their reply to my question.
9. L. 195. I disagree that 12 months of storage are a time zero of the screening. This should be instead the very beginning of the storage experiments once seeds have been fully equilibrated under the targeted RH.
10. L 196 RH and not HR.
11. L. 197-204 All statement here is missing any statistical support. Why not running these statistical comparisons through one or >1 ANOVAs or non-parametric tests? Despite the main objective of “provide and overall interpretation of the data analysis”, data analysis requires statistical support as the comparisons to which the authors draw attention do.
12. L. 246 “Enhanced” not enhance.
13. L. 246-256 As for point 11. All the statements miss statistical evidence.
14. L 256 “estimated humidity values” were decreased. This is very unclear and confusing to me. What are the authors referring to? RH values in the chamber? If so, they declared that they were set at 25 and 45% RH (Table 2) and do not match with values in the figures. Or are they talking about seed moisture contents? This point requires maximal attention.
15. L. 300 This may indicate that..”…”. It is unclear what this is referring to. Rephrase needed.
16. L 306 Contributed not “contribute”.
17. L. dark green (there is a typo).
Discussion
L. 323 Probably, it is better to write about ROS “production” rather than ROS accumulation.
L. 328 A comma is missing after [17] and before “makes”.
L. 330 Lipid peroxidation and (instead of comma) and reduced levels of antioxidants. A supporting reference is also missing at the end of this statement.
L. 339-341. Why? I could not see a clear rationale behind the choice of the storage intervals. Do authors have any previous evidence (from previous published or unpublished work) the guided the choice of 12 and 22 months? One would intuitively expect that seed longevity will decline faster at 25°C and 45% RH rather than other ageing regimes.
L. 350 Please, type in a full stop between the words “storage” and “however”.
L. 355. State “ROS and RNS include…” instead of “among them, are…”.
L. 362 Probably it would be clearer to talk about seed coat “macromolecular composition” rather than seed coat “environment”.
L. 364-366. Same as for points 11 and 13. This conclusion is not statistically supported.
L. 375. Replace “seed germination in terms of storability” with seed longevity.
L. 384 Please, refer to seed moisture content rather than “desiccant”. Moreover, the gaseous composition of storage atmosphere (e.g., control of oxygen) is another important factor that would benefit of more studies and implementation at the industrial scale.
Conclusions
L. 388 Use “seed storability” rather than “high sensitivity to storage”.
Figures and captions
The resolution of the figure is poor and was better in the previous submission. Figures appear quite blurred.
Table 2. Exact values of temperatures are needed in case of <10°C. This is too general, given the influence of storage temperatures on longevity. Authors should at least provide a range. Regarding seed water or moisture content, this critical to seed storage as much as the temperature conditions under any storage regimes, including standard commercial storage.
Figures 1. In the caption, Welsh should always be capitalised.
Figures 2 and 3. Error bars were added but without specifying what they stand for. SE or SD? It would be good to also mention the number of replicates in the caption. My remark on the conceptual mistake of connecting categorical variables with lines (ie, ROS and RH) was disregarded.
In the caption, authors should spell out all abbreviations at first use (ROS, RH – missing)
Tables 3 and 4. The term “P-values” Persons’s “r” are not consistently italicised.
Figure 4. The font of the legend in panel b remained too small and hard to read. Use “RH” instead of “humid” also in the figures for consistency and ease of interpretation. Once more, I recommend specify to what P-values are referring in panel a (I assume r coefficients).
In summary, I encourage the authors to: i) conduct statistical tests to support the statements in their results; ii) change the type of representation of ROS and RH values in the figures (additional error bars could be an option); iii) clarify the meaning of RH values measured during the storage intervals. In my view, these are essential pending amendments to warrant publication of the manuscript.
Yours sincerely
Comments on the Quality of English LanguageMinor typos and a couple of sentences with a structure that can be improved. See punctual comments from previous field.
Author Response
The manuscript has been revised, changes are highlighted in red.
We hope we have fully addressed the Reviewers' requirements
best regards
Alma Balestrazzi
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed all the comments. However, the quality of the figures is still not up to the mark.
Comments on the Quality of English LanguageModerate editing of English language is required.
Author Response
We thank the reviewer. Changes in the text are highlighted in red.
Author Response File: Author Response.pdf
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsPlease see the attached file.
Comments for author File: Comments.pdf
Author Response
We thank the Reviewer for his final acceptance
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors considered most of the suggestions and requests from earlier assessment of the manuscript. Importantly, they conducted statistical analyses to support some of their findings and clarified on previously reported RH values used for commercial storage of seed lots. However, the draft contains aspects that deserve further attention. Note that I could not find any supplemental material in the attachments (ANOVA tables, etc...), although this would not substantially change my decision. Some conclusions do not appear to be statistically supported. Additional comments to be addressed are listed here as follows:
L. 25-26 “this study has also evidenced that”. Please, remove. It is redundant.
L. 99 Amend to “at 25 and 45%”, as they appear to be the only two values. There was no range. Furthermore, I warmly recommend seed moisture content and/or equilibrium relative humidity of seeds in any future study. This can be done by drying in an oven (17 h, 103°C) and using a hygrometer, respectively.
Table 1. Genetic background of BO13a was missing.
l. 169 Where are these tables?
L. 170 Remove superscript. There were no superscript letters. Use consistently “P” or “p-value” through the manuscript, following the journal’s guidelines.
L. 182 “seed lots“ instead of samples.
L. 184 Use “seed lots” instead of “variety” throughout. I think this would help avoid any confusion.
L. 186 Rephrase as “between ROS level and seed germination, as a measure of viability”.
L. 187-189 Guidance needed here. The code in Table 3 suggested me that 1 seed lot only was assessed (1801030128 – BO14a, as from table 1). Was this really the case? If so, why? The same applies to Table 4.
Fig. 2 and 3. Once more, I ask the author to change the representation of ROS levels in all panels. Mine is not a merely aesthetic reason but a relevant formal aspect: categorical variables cannot be connected be lines. ROS values could be either presented as another series of bar or a scatterplot. IN tha captions, remove “superscript” and “as analyzed by one-way ANOVA and Tukey-Kramer test”, as it is already mentioned earlier.
L. 313-315 All repetitive, can be deleted.
L. 215-216 These conclusions on BO12 samples were not supported by the statistical analyses.
L. 216-218 “Thus, …, consideration”. This seems to contradict previous statement on condition F.
L. 219 Amend the typo in the word “between”.
L. 256-257. This did not seem to be the case for BO06 and BO13. There are indeed no asterisks in Fig. 4.
L. 261 Rephrase as “the highest impact of storage conditions on seed germination, expressed as log2 (germ22/germ12)” and “was recorded” (past tense needed here).
Fig. 4. In the caption, change to “observed between 12 and 22 months of storage” and “assessed after”, not “assessed at”. Furthermore, the caption missed information on the storage conditions. At least a reference to Table 2 should be included to guide readers and make the figure a self-standing element.
L. 276 I am confused here. What was meant by “complete correlation”? What is the difference now with data shown in Tables 3 and 4.
L. 277 Use “storage intervals” instead of “timepoints of sampling analyses”. I believe that PCA requires spelling out at first use.
L. 284 71.2% Where was this value coming from? This is different from PC1+PC2. Please, clarify.
L. 285-290 The past tense needs to be used consistently here. There is a typo for dark green.
Figure 5. Caption. The closer the value is to ±1. The value of what? Clarify. Moreover, the storage conditions should be clearly referenced by referring to Table 2.
L. 304-305 This sentence requires toning down. Lipid peroxidation and consumption of antioxidants are common but not the rule.
L. 305-306. This depends very much on the seed moisture contents that essentially dictate the temperature range for storage. Please, mention seed moisture contents as well.
L. 312 Mention storage conditions as defined in the paper (temperature, seed moisture content and % oxygen)
L. 312-313 This is a relevant information that should be given earlier in the results. Nonetheless, I missed some information here: how often was germination monitored during the first 12 months? Why were ROS levels not measured immediately before any storage, at least?
L. 315-317 “In our opinion, ,,,, storability”. I still do not quite follow the logic here. Please, argue better or leave the sentence out.
L. 319-320 Help identify (not identifying).
L. 342 Use “certain” rather than “specific”.
L. 343. It would have been much more informative to measure equilibrium relative humidity (eRH, sometimes referred to water activity) and seed moisture content.
L. 355 “Conducted” instead of “investigated”.
L. 356-357 “however… discussed”. Delete. It is just wordy.
L. 360 I think the paper was misreferenced here.
L. 361 What make an assay “smart” then? The authors should use a more appropriate term or argue better this point. In addition, I do not see any contrast between the two sentences. Why but? Please, revise.
L. 368 Specify poor storability.
L. 372 Provide examples of which parameters should be screened. It read too general to me.
L. 373-374 Extensive literature shows that most of this variability is indeed imposed by the parental environment, which is reflected by initial quality of a seed lot prior to any storage. Once more, I invite the authors to be clearer in their vision here. Did they mean that screening seed lots would ideally lead to disregard those lots with poor storability prior to any storage? Please, clarify.
Overall, the quality of the figures remained poor or very poor. I leave this to the editorial office to check.
In summary, I am afraid, but the manuscript requires further attention before my recommendation to publishing.
Kind regards
Comments on the Quality of English LanguageSome typos and misuse of tenses were remarked in the comments.
Author Response
Please, see the attachment
Author Response File: Author Response.pdf
