Tissue-Specific Transcriptomes Outline Halophyte Adaptive Strategies in the Gray Mangrove (Avicennia marina)

Round 1

Reviewer 1 Report

The article entitled “Tissue-specific transcriptomes outline halophyte adaptive strategies in the gray mangrove, Avicennia marina” has been an excellent study to explore the tissue specific transcriptome and that too from different locations. But my observations include as below:

1.      In general, English language and sentence making need revision.

2.      The purpose of studying transcriptomes from different tissues and geographies is not clear in the introduction. Include with clear objectives.

3.      In materials and methods, the authors have mentioned the source of RNA isolation. It would be better if the authors briefly give details of the conditions of plant growth and environment from where they isolated RNA of different tissues. Additionally, the physio-chemical properties of locations w.r.t saline/stress levels will further clarify the sample differences.

4.      Line 88-89: Sentence seems to be incomplete

5.      Line 94-95: tissue-specific transcriptomes to natural and treatment-induced gene expression patterns. Here, what is the treatment?

6.      In Results, there is no p-value of DEGs?? The physiological data similarity and Quantitative functional validation of some halophytic genes would further enrich the data.

7.      The Discussion needs to emphasize the novelty of these results and to explain more clearly how they represent an advance on previous studies. Authors did not mention well to the previous studies that deal with the same topic, especially the newest one. Moreover, at the end of this section, please illustrate what hypothesis this investigation aimed to test.

8.      Pictorial interpretation of results is good but clearly differentiate between the transcriptomic analysis of different locations. Also, highlight the specific genes for particular traits/process from each sample as well as location e.g. if DEGs for photosynthesis or antioxidants, what is there expression level w.r.t all collection sites and each plant part.

9.      In conclusion, briefly summarize which genes are most prominent and which are conserved throughout different locations.

Author Response

REVIEWER 1 – Author responses

The article entitled “Tissue-specific transcriptomes outline halophyte adaptive strategies in the gray mangrove, Avicennia marina” has been an excellent study to explore the tissue specific transcriptome and that too from different locations. But my observations include as below:

1. In general, English language and sentence making need revision.

RESPONSE: English language was revised where necessary.

2. The purpose of studying transcriptomes from different tissues and geographies is not clear in the introduction. Include with clear objectives.

RESPONSE: We have added text (lines 106-115) to address the reviewer’s comment. We have outlined our objectives in the abstract (lines 16-20): “We compare transcriptomes from A. marina pneumatophores, stems, leaves, flowers, seeds, and transcriptomes across four widely divergent environments in the Indo-Pacific (Red Sea, Arabian Gulf, Bay of Bengal, Red River Delta) to decipher shared and location-, tissue- and condition-specific functions.”

3. In materials and methods, the authors have mentioned the source of RNA isolation. It would be better if the authors briefly give details of the conditions of plant growth and environment from where they isolated RNA of different tissues. Additionally, the physio-chemical properties of locations w.r.t saline/stress levels will further clarify the sample differences.

RESPONSE: The sources of the RNAseq data were described in our original submission in section 2.1. ‘Sequence sources’, lines 85-95.

4. Line 88-89: Sentence seems to be incomplete

RESPONSE: The original sentence read: “Reference transcripts for expression analyses were from [7]” to indicate the reference for the source. The sentence has been modified to now state “Reference transcripts for expression analyses were from reference He et al., 2020 [7]” for clarity.

5. Line 94-95: tissue-specific transcriptomes to natural and treatment-induced gene expression patterns. Here, what is the treatment?

RESPONSE: The phrase ‘and treatment-induced’ was incorrect and removed.

6. In Results, there is no p-value of DEGs?? The physiological data similarity and Quantitative functional validation of some halophytic genes would further enrich the data.

RESPONSE: No p-values were required for the analyses we performed on the transcriptomic profiles, except for enrichment analyses and those were presented on line 154: “A false discovery rate (FDR) threshold of 0.001 was used for detection of enrichment”. Note that we have changed all instances of “FDR P = “ to the correct format of “FDR p = ” . Quantitative functional validation is outside the scope of this study.

7. The Discussion needs to emphasize the novelty of these results and to explain more clearly how they represent an advance on previous studies. Authors did not mention well to the previous studies that deal with the same topic, especially the newest one. Moreover, at the end of this section, please illustrate what hypothesis this investigation aimed to test.

RESPONSE: We have revised the Discussion to address the reviewer’s comments (see lines 677-696). We have added several references to recent work; see references [1-7] in the bibliography for this response letter.

8. Pictorial interpretation of results is good but clearly differentiate between the transcriptomic analysis of different locations. Also, highlight the specific genes for particular traits/process from each sample as well as location e.g. if DEGs for photosynthesis or antioxidants, what is there expression level w.r.t all collection sites and each plant part.

RESPONSE: Transcriptomic analyses of different locations was defined in its section starting on line 569 titled “3.8. Comparison of A. marinatranscriptomes across geographies and climates”. The different locations are also explained in the Fig. 5 legend as well as in section 2.1. ‘Sequence sources’. All the data requested by Reviewer 1 was in the supplemental material that was provided to the reviewers (e.g., see Table S1).

9. In conclusion, briefly summarize which genes are most prominent and which are conserved throughout different locations.

RESPONSE: We have added text and rewritten the conclusion section to address the reviewer’s comment (see lines 801-813).

References

1. Rubinovich, L.; Weiss, D. The Arabidopsis cysteine-rich protein GASA4 promotes GA responses and exhibits redox activity in bacteria and in planta. The Plant journal : for cell and molecular biology 2010, 64, 1018-1027, doi:10.1111/j.1365-313X.2010.04390.x.
2. Roxrud, I.; Lid, S.E.; Fletcher, J.C.; Schmidt, E.D.; Opsahl-Sorteberg, H.G. GASA4, one of the 14-member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development. Plant Cell Physiol 2007, 48, 471-483, doi:10.1093/pcp/pcm016.
3. Aubert, D.; Chevillard, M.; Dorne, A.M.; Arlaud, G.; Herzog, M. Expression patterns of GASA genes in Arabidopsis thaliana: the GASA4 gene is up-regulated by gibberellins in meristematic regions. Plant molecular biology 1998, 36, 871-883, doi:10.1023/a:1005938624418.
4. Salekeen, R.; Mou, S.N.; Islam, M.E.; Ahmed, A.; Billah, M.M.; Rahman, S.M.M.; Islam, K.M.D. Predicting multi-enzyme inhibition in the arachidonic acid metabolic network by Heritiera fomes extracts. J Biomol Struct Dyn 2022, 40, 4259-4272, doi:10.1080/07391102.2020.1855248.
5. Hanin, M.; Brini, F.; Ebel, C.; Toda, Y.; Takeda, S.; Masmoudi, K. Plant dehydrins and stress tolerance: versatile proteins for complex mechanisms. Plant signaling & behavior 2011, 6, 1503-1509, doi:10.4161/psb.6.10.17088.
6. Natarajan, P.; Murugesan, A.K.; Govindan, G.; Gopalakrishnan, A.; Kumar, R.; Duraisamy, P.; Balaji, R.; Tanuja; Shyamli, P.S.; Parida, A.K.; et al. A reference-grade genome identifies salt-tolerance genes from the salt-secreting mangrove species Avicennia marina. Commun Biol 2021, 4, 851, doi:10.1038/s42003-021-02384-8.
7. Pandey, B.K.; Mehra, P.; Verma, L.; Bhadouria, J.; Giri, J. OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation. Plant physiology 2017, 174, 2316-2332, doi:10.1104/pp.17.00571

Author Response File: Author Response.docx

Reviewer 2 Report

 The authors compared different tissues of Avicennia marina from different places to get the tissue-specific genes and the transcriptional profile in divergent environments. Compared with other inland species, Avicenniashows a high gene expression level in heat and drought tolerance. The flower tissue-specific genes enrich in nectar formation pathway, which is important for plants’ pollination. Seed-specific expressed genes regulate seed senescence and ripening timing. Leaf tissue specific genes are related to photosynthesis, heat and salt responses. In pneumatophore, the tissue-specific genes are related to hypoxia and salt responses. The topic is foundational for mangrove studies, and the result is interesting to specific audience.

However, my enthusiasm was dramatically reduced for the result analysis. They mostly use language description on high and low gene expression are; these are very difficult to read and draw conclusions from.  These should be organized into a table or graphic presentations. While it is with rich supplemental data, they are lists of raw results yet to be extracted, analyzed, and presented. 

The most important point is that how these findings related to Agronomy – improving crop production and practices?  While there are potential implications, it is far from a reference to crop improvement.  It seems the paper is more suitable for ecological journals. 

It is also called to questions on how comparable of these datasets are.  They are from different geographical locations, different time and environmental conditions and methods of RNA-seq library production and sequencing.

Other comments

1.    In the abstract, it would be better to summarize the tissue-specific genes of each tissue. The authors only mentioned flower and leave. The authors can pay more effort to some transcriptional profiles that inland plants do not have, e.g. pneumatophore tissue-specific genes related to hypoxia and heat responses, leaf tissue related to heat and salt responses.

2.    The introduction needs to be reorganized. From the introduction I can not understand why the authors want to study the tissue specific transcriptomes. It did not highlight the research needs and importance of studying tissue specific issue in Avicennia over different ranges. 

3.    The paper analyzed the transcription of pneumatophore but called the tissue specific genes as “root specific genes”. It is not quite accurate, and it is suggested to change “root” to “pneumatophore”. It would be better to descript the tissues in the method, e.g., the pneumatophore samples were collected the pneumatophore tips.

4.    UMAP is powerful for clustering identity unknown cells. In line 126-128, the authors use UMAP to cluster tissues and annotate different tissues, but they already know which tissue they are testing. Compared with testing the specific tissue, the clustering results may not be accurate. 

5.    Fig 2, UMAP does not show 3D clearly. These right side panels do not offer enough information.

6.    In methods, the parameters for filtering significant DEGs, log2FC and adjusted P-value should be given.

Minor comments

1.    It would be more helpful to show the PCA results in supplementary. 

2.    The study obtained transcription raw data from different studies and need to remove the batch effect before calculating DEGs.

3.    In line 353, check the format mistake.

4.    It seems the format for references is not consistent, the authors should check before publishing.

5.    In number of locations, “Lignan” should be “Lingnan” Normal Univeristy

6.    Line 172, “Shi lab reference transcripts” need citation 

7.    Line 242, “One of the most remarkable findings from our studies” but the results are not shown in the main text.

8.    Fig 5, the violine plot needs axis. 

Author Response

REVIEWER 2 – Author responses

R2 (gray):  The authors compared different tissues of Avicennia marina from different places to get the tissue-specific genes and the transcriptional profile in divergent environments. Compared with other inland species, Avicenniashows a high gene expression level in heat and drought tolerance. The flower tissue-specific genes enrich in nectar formation pathway, which is important for plants’ pollination. Seed-specific expressed genes regulate seed senescence and ripening timing. Leaf tissue specific genes are related to photosynthesis, heat and salt responses. In pneumatophore, the tissue-specific genes are related to hypoxia and salt responses. The topic is foundational for mangrove studies, and the result is interesting to specific audience.

However, my enthusiasm was dramatically reduced for the result analysis. They mostly use language description on high and low gene expression are; these are very difficult to read and draw conclusions from.  These should be organized into a table or graphic presentations. While it is with rich supplemental data, they are lists of raw results yet to be extracted, analyzed, and presented. 

The most important point is that how these findings related to Agronomy – improving crop production and practices?  While there are potential implications, it is far from a reference to crop improvement.  It seems the paper is more suitable for ecological journals. 

RESPONSE: The suggestion is well received. Critical links between the topics were elaborated upon in the revised manuscript. In our original submitted manuscript, we presented and described halophyte transcriptomics with the aim to inform saline agriculture.

We have added text in several locations throughout the manuscript to address the reviewer’s comment (lines 16-17, 106-115, 564-569, 615-622, 790-799, 801-813).

Please note that our initial manuscript was transferred to this issue on the recommendation of the journal editors.

It is also called to questions on how comparable of these datasets are.  They are from different geographical locations, different time and environmental conditions and methods of RNA-seq library production and sequencing. 

RESPONSE: Use of the TPM metric was developed to compare across samples with different origin methods and locations. For example, in the Genome Biology paper ‘A survey of best practices for RNA-seq data analysis’, the authors state “TPMs, which effectively normalize for the differences in composition of the transcripts in the denominator rather than simply dividing by the number of reads in the library, are considered more comparable between samples of different origins and composition” (Conesa et al., 2016).

Other comments

1. In the abstract, it would be better to summarize the tissue-specific genes of each tissue. The authors only mentioned flower and leave. The authors can pay more effort to some transcriptional profiles that inland plants do not have, e.g. pneumatophore tissue-specific genes related to hypoxia and heat responses, leaf tissue related to heat and salt responses.

RESPONSE: Stems were also mentioned, but the reviewer has a good point. We have revised the abstract to mention features of each of the five tissues in the manner recommended by the reviewer (lines 23-38).

2. The introduction needs to be reorganized. From the introduction I can not understand why the authors want to study the tissue specific transcriptomes. It did not highlight the research needs and importance of studying tissue specific issue in Avicennia over different ranges. 

RESPONSE: The introduction was reorganized to explain how the tissue and region-specific transcriptomes hold valuable information about halophyte biology that can expedite and inform saline agricultural efforts.

3. The paper analyzed the transcription of pneumatophore but called the tissue specific genes as “root specific genes”. It is not quite accurate, and it is suggested to change “root” to “pneumatophore”. It would be better to descript the tissues in the method, e.g., the pneumatophore samples were collected the pneumatophore tips.

RESPONSE: We have changed the wording from ‘roots’ to ‘pneumatophores’ where appropriate for clarification. The reviewer is invited to review our data source disclosures in the Materials and Methods as well as in the Data Availability Statement.

4. UMAP is powerful for clustering identity unknown cells. In line 126-128, the authors use UMAP to cluster tissues and annotate different tissues, but they already know which tissue they are testing. Compared with testing the specific tissue, the clustering results may not be accurate. 

RESPONSE: UMAP was done to see how tissues group together, as stated in lines 271 to 278 of the original text.

5. Fig 2, UMAP does not show 3D clearly. These right side panels do not offer enough information.

RESPONSE: The comment is well-received. We have summarized the information presented in the original panels as a bubble plot showing unique KEGG metabolic pathways for each tissue. This is the new Fig. 3. The previous Fig. 3 is the current Fig. 2.

6. In methods, the parameters for filtering significant DEGs, log2FC and adjusted P-value should be given.

RESPONSE: These statistics are not relevant to the analyses that we performed. See lines 153-163 for the description of the methods we used.

Minor comments

1. It would be more helpful to show the PCA results in supplementary. 

RESPONSE: We never presented PCA results in the original submission. Does the reviewer mean the UMAP results? If the reviewer is referring to the UMAP graph, that has been moved to the supplementary materials to accommodate the new Fig. 3.  

2. The study obtained transcription raw data from different studies and need to remove the batch effect before calculating DEGs.

RESPONSE: Removal of the batch effect was inherent in our sampling technique. See lines 153-163 for the description of the methods we used.

3. In line 353, check the format mistake.

RESPONSE: We would appreciate it if the reviewer could specify the mistake.

3. It seems the format for references is not consistent, the authors should check before publishing.

RESPONSE: The reference style configuration was downloaded from https://endnote.com/style_download/mdpi/ and applied to the revised manuscript.

5. In number of locations, “Lignan” should be “Lingnan” Normal Univeristy

RESPONSE: In our original submission, every instance of ‘Lignan’ was already stated as “Lingnan Normal University”.

6. Line 172, “Shi lab reference transcripts” need citation 

RESPONSE: A citation was added.

7. Line 242, “One of the most remarkable findings from our studies” but the results are not shown in the main text. 

RESPONSE: The findings were indeed mentioned throughout the main text (see lines 33-34, 465-469, 489-491, 553-563)

8. Fig 5, the violine plot needs axis. 

RESPONSE: Writing was added to clarify that the Y-axis is for both plots in the Fig. 5 legend (lines 678-679): “Expression values for A. marinaleaves from the Red Sea and India, Shanghai, and Vietnam coasts, as well as their distributions, are shown along the y-axis”.

REFERENCES

Conesa, A., Madrigal, P., Tarazona, S., Gomez-Cabrero, D., Cervera, A., McPherson, A., Szczesniak, M.W., Gaffney, D.J., Elo, L.L., Zhang, X. and Mortazavi, A.  2016.  A survey of best practices for RNA-seq data analysis. Genome Biol 17, 13.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors compared transcriptomes from Avicennia marina roots, stems, leaves, flowers, seeds, and transcriptomes across four widely divergent environments in the Indo-Pacific (Red Sea, Arabian Gulf, Bay of Bengal, Red River Delta) to decipher shared and location-, tissue- and condition-specific functions. The work is relevant, timely and presents new data. The study is well structured and the results is well presented.

 However, here is some places in the paper that require adjustments:

 Line 3. In the title it is better to put the Latin name in brackets …… in the gray mangrove (Avicennia marina)

Line 32. In keywords Gray mangrove is repeated twice. Remove one of them.

Check if the list of references is formatted according to the requirements of the journal. 

Best wishes.

Author Response

Reviewer 3 - Authors - Responses

R3 (gray) The authors compared transcriptomes from Avicennia marina roots, stems, leaves, flowers, seeds, and transcriptomes across four widely divergent environments in the Indo-Pacific (Red Sea, Arabian Gulf, Bay of Bengal, Red River Delta) to decipher shared and location-, tissue- and condition-specific functions. The work is relevant, timely and presents new data. The study is well structured and the results is well presented. 

 However, here is some places in the paper that require adjustments:

 Line 3. In the title it is better to put the Latin name in brackets …… in the gray mangrove (Avicennia marina)

RESPONSE: The suggested change was made to place the Latin name in brackets in the title.

Line 32. In keywords Gray mangrove is repeated twice. Remove one of them.

RESPONSE: The British spelling ‘Grey mangrove’ was removed

Check if the list of references is formatted according to the requirements of the journal. 

Best wishes.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript has been improved a lot.  But the following issues remain.

1. Simple search found 4 "Lignan", lines 110, 192, 233, 741.  These must be corrected to "Lingnan" please! Because this is the name. 

2. There are continue issues with the references.  There are 168 references for such a paper.  It seems it's more than a review paper. Is this the policy of the journal?  Moreover, there are so many unknown "%U" in many lines. Please check.

Author Response

The manuscript has been improved a lot.  But the following issues remain.

1. Simple search found 4 "Lignan", lines 110, 192, 233, 741.  These must be corrected to "Lingnan" please! Because this is the name. 

            RESPONSE: The spelling was corrected to Lingnan

2. There are continue issues with the references.  There are 168 references for such a paper.  It seems it's more than a review paper. Is this the policy of the journal?  Moreover, there are so many unknown "%U" in many lines. Please check.

RESPONSE: The “%U” was an artifact from Endnote and all instances of “%U” have been deleted.