The Aquaporin Gene SbPIP1;2 Is Involved in Dormancy Release and Regulated Under Low Temperatures in Lilium ‘Siberia’

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript studies the role of the aquaporin gene SbPIP1;2 in dormancy release and flower bud development in lily bulbs stored at low temperature. The topic is interesting and relevant, especially for understanding dormancy mechanisms in ornamental plants. The combination of VIGS, transcriptome analysis, hormone measurements, and morphological observations is valuable, and the results suggest that SbPIP1;2 may play an important role during dormancy release. The manuscript is generally well organized, and the figures are clear. However, several points should be improved before publication. Below are my comments:

1) The title is slightly too strong compared to the presented results.

2) The aim of the study is understandable, but the research hypothesis is not clearly stated in the Introduction.

3) Some methodological details are unclear:

- the exact number of biological replicates was not clearly provided for all analyses,

- the statistical analysis was not described clearly enough,

- the number of bulbs used in the experiments was not precisely explained,

- the description of the VIGS method is sometimes unclear and difficult to follow,

- information about the analysis of hormone data is missing,

- the qRT-PCR validation is described only briefly.

4) Results: the transcriptome analysis identified many differentially expressed genes, but I think that additional validation of several important genes by qRT-PCR would be valuable.

5) Discussion: the study shows that silencing SbPIP1;2 delays dormancy release, but this mechanism is still not fully explained. I suggest that the discussion should better explain the relationship between SbPIP1;2, hormone changes, and carbohydrate metabolism.

6) Discussion: some parts of the Discussion are too general and repeat information already known from the literature. The authors should focus more on interpreting their own results.

7) Gene names and abbreviations should be formatted more consistently.

Comments on the Quality of English Language

The manuscript requires English language editing. There are many grammatical and stylistic problems throughout the text, as well as several typing mistakes.

Author Response

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Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have satisfactorily implemented the requested modifications, and the manuscript is now suitable for publication.

Author Response

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Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

This work focus on the Lily's PIP function with noble physiological contribution. Very interesting work but needs some attention as following.

1. Introduce how the lily's bud germinates and how are the cells in those tissues contribute.
2. The evidence to believe PIP1;2 is not inside the nucleus is required. e.g. is there any picture for PIP1;2-GFP with hallow nucleus or such? OR, provide multiple pictures showing no signal near nucleus-like structure but only in cytosol.
3. Is there any picture of PIP1;2 silenced flower structure? It would definitely increase your data's credibility.
4. It is confusing to see PIP1;2 function in floral bud and bulbs. Is it possible to focus and show? (it is okay when the silenced lines does not show any phenotype) OR, show both 'silenced' and 'overexpressed' lines for both functions.
5. Using transcriptome for the PIP1;2 function is not an usual approach when it is an aquaporin protein. Is there any reason?
6. Full genome for 'Lilium sargentiae' is published and available. Also, the Liang et al., Nature Comm. 2025 showed that siberia RNA-seq was able to be analyzed with L. sargentiae. Provide valid reason for assembling transcriptome or re-analyze with L. sargentiae.

Author Response

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Round 2

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

I can see the authors were sincerely assessing the revision, yet some portion still bothers a lot.

1. Revised supplementary figure 2 was not including confocal image. You have to show the data for your scientific mention.
2. On the transcriptome analysis and assembly, the response of authors are not satisfactory. Followings are the details.
   1. Without the reference genome information, you can not target the gene to study. Or, at least there should be an introductory data for why the gene was chosen when there are a lot of PIPs.
   2. The reason we use reference genome is not just because we want to perform the genomic analysis but to be 'sure' whether the gene information is true. Thus, either you show your assembly is complete (maybe with BUSCO) or using the reference genome is DEFINITELY required.

Author Response

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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

Comments and Suggestions for Authors

It is always interesting to see the noble molecular biological works in the non-model system. If it goes successful, it gives a great influence on the botanical community. However, I found some of the points are requiring the author's focus as following. Though the points are few, it bothers me to 'believe' or 'understand' all of your works.

1. Which gene did you cloned? In the legend of Figure S1, you said it was PIP2;3 however in the main text it was PIP1;2.
2. Your cloned gene localized in the nucleus even though it is an aquaporin family. Was there any NLS domain or any other explanation for this? If this is true in vivo, it would be a good discovery however for now, I cannot believe this. Please confirm your cloned vector and transformant.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

see PDF

Comments for author File: Comments.pdf

Comments on the Quality of English Language

SEE PDF

Author Response

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Author Response File: Author Response.pdf