Genome-wide Identification and Expression Analysis of TALE Gene Family in Pomegranate (Punica granatum L.)
Round 1
Reviewer 1 Report
In the manuscript "Genome-wide identification and expression analysis of TALE gene family in pomegranate (Punica granatum L.)", the authors tried to identify TALE superfamily genes in pomegranate based on gene homology. They identified 16 PgTALE genes via bioinformatics methods. The authors explored their phylogenetic relationships. They also found that the PgTALE gene structure of the subfamily is very similar. Function prediction and protein-protein network analysis showed that PgTALE might participate in regulating the development of apical meristems, flowers, carpels, and ovules. Analysis of gene expression patterns showed that the PgTALE genes had a particular tissue expression specificity. The authors said that these conclusions were the foundation for the function research of the PgTALE gene and provided a reference for exploring its evolutionary process, and that the knowledge in pomegranate might be applied to other fruit as well.
In general, the data presented in the manuscript are interesting. However, several points need to be addressed to improve the manuscript:
1, “TALE” can be short for "Transcription activator-like effector" or "three-amino-acid-loop-extension". The latter one seems to be the correct one. The authors should give the full name for "TALE" in the manuscript.
2, In the manuscript, the authors did not give the full names for most of the abbreviations. For example: TALE, BELL, KNOX, all the abbreviations in line 34-39. If possible, the authors should provide a list with abbreviations and their full names.
3, The author should provide the information that pomegranate and Eucalyptus grandis are in the same Order: Myrtales. Otherwise, the readers may not know why they chose E. grandis to construct the phylogenetic tree. I chose “Can be improved” for “Does the introduction provide sufficient background and include all relevant references?”. The authors should provide more background about E. grandis and the Myrtales Order.
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a point-by-point response to each comments.
Point 1: "TALE" can be short for "Transcription activator-like effector" or "three-amino-acid-loop-extension". The latter one seems to be the correct one. The authors should give the full name for "TALE" in the manuscript.
Response 1: "TALE" can be short for "three-amino-acid-loop-extension", we have revised this paper based on your suggestion.
Point 2: In the manuscript, the authors did not give the full names for most of the abbreviations. For example: TALE, BELL, KNOX, all the abbreviations in line 34-39. If possible, the authors should provide a list with abbreviations and their full names.
Response 2: The full name of "TALE" is "three-amino-acid-loop-extension", "BELL" is "BEL1-like"," KNOX" is " KNOTTED-like homeobox " and etc. We have revised this paper based on your suggestion and provided a list with abbreviations and their full names in the supplements.
Point 3: The author should provide the information that pomegranate and Eucalyptus grandis are in the same Order: Myrtales. Otherwise, the readers may not know why they chose E. grandis to construct the phylogenetic tree. I chose "Can be improved" for “Does the introduction provide sufficient background and include all relevant references?”. The authors should provide more background about E. grandis and the Myrtales Order.
Response 3: Thanks, we modified as "Myrtales, the myrtle order of flowering plants, is placed in the Angiosperm Phylogeny Group IV (APG IV) botanical classification system [37]. Pomegranate (Punica granatum L.) is a considerable economic fruit tree of the Lythraceae family and widely cultivated worldwide, in addition, it was that pomegranate and the related species Eucalyptus grandis H., belonging to the order Myrtales, shared the paleotetraploidy event [38] ".
- Byng, J.W.; Chase M.W.; Christenhusz, M.J.M.; Fay, M.F.; Judd, W.S.; Mabberley, D.J.; Sennikov, A.N.; Soltis, D.E.; Soltis, P.S.; Stevens, P.F.; Briggs, B.; Brockington, S.; Chautems, A.; Clark, J.C.; Conran, J.; Haston E.; Moller, M.; Moore, M.; Olmstead, R.; Perret, M.; Skog, L.; Smith, J.; Tank, D.; Vorontsova, M.; Weber, A. An update of the angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot. J. Linn. Soc. 2016, 181, 1-20, doi:10.1111/boj.12385.
- Yuan, Z.; Fang, Y.; Zhang, T.; Fei, Z.; Han, F.; Liu, C.; Liu, M.; Xiao, W.; Zhang, W.; Wu, S. The pomegranate (Punica granatum L.) genome provides insights into fruit quality and ovule developmental biology. Plant Biotechnol. J. 2018, 16, 1363-1374, doi:10.1111/pbi.128
Note: In addition, A. thaliana contains a member of the TALE gene family KNATM without homeodomain, we added a pomegranate gene homologous to KNATM [32,33]. Therefore, a total of 17 genes (not 16) of pomegranate TALE family were identified and analyzed in pomegranate, we have revised our manuscript, including contents and figures.
- Magnani, E., Hake, S. KNOX lost the OX: the Arabidopsis KNATM gene defines a novel class of KNOX transcriptional regulators missing the homeodomain. Plant Cell 2008, 20, 875-887, doi: 10.1105/tpc.108.058495.
33. Hamant, O., Pautot V. Plant development: A TALE story. C. R. Biologies 2010, 333, 371-381, doi: doi.org/10.1016/j.crvi.2010.01.015
Reviewer 2 Report
The manuscript is an interesting approach to the identification and expression analysis of TALE genes in pomegranate. In general, the manuscript is well designed and written.
However, I suggest some small changes to the authors.
The nomenclature of the genes should be revised. The name of genes should write in italic through the text.
For the readers, could be adequate to clarify the name of the genes when they are named the first time.
The size of the Figure 2 and 5 should be modified. In current scale is very difficult to see the showed data.
L 284-287. The Latin names of these species should be completed with the authors.
L 329: I don’t understand this. All variations in any gene are due to ‘the expansion and evolution of the PgTALE gene’, independently these changes generated gene or pseudogene variants. This could be deleted.
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a point-by-point response to each comments.
Point 1: The nomenclature of the genes should be revised. The name of genes should write in italic through the text.
Response 1: We have revised the nomenclature of the genes and written the name of genes in italic through the text.
Point 2: For the readers, could be adequate to clarify the name of the genes when they are named the first time.
Response 2: Thanks, we clarified the name of the genes when they were named the first time, and provided a list with abbreviations and their full names in the supplements.
Point 3: The size of the Figure 2 and 5 should be modified. In current scale is very difficult to see the showed data.
Response 3: Based on your suggestion, we modified the size of the Figure 2 and 5.
Point 4: L 284-287. The Latin names of these species should be completed with the authors.
Response 4: We added the authors of the Latin names of these species based on your suggestion. We modified as ‘Currently, the TALE gene family has been found in many plants: 33 AtTALE genes in A. thaliana, 40 LjTALE genes in Lotus japonicas K. [61], 46 GaTALE genes in Gossypium arboretum L., 47 GrTALE genes in G. raimondii L., 88 GbTALE genes in G. barbadense L., 94 GhTALE genes G. hirsutum L. [8], and 35 PtTALE genes in poplar [62], 7 VsTALE genes in Vandenboschia speciose G. [16].’
Point 5: L 329: I don’t understand this. All variations in any gene are due to ‘the expansion and evolution of the PgTALE gene’, independently these changes generated gene or pseudogene variants. This could be deleted.
Response 5: We deleted ‘the expansion and evolution of the PgTALE gene’.
Note: In addition, A. thaliana contains a member of the TALE gene family KNATM without homeodomain, we added a pomegranate gene homologous to KNATM [32,33]. Therefore, a total of 17 genes (not 16) of pomegranate TALE family were identified and analyzed in pomegranate, we have revised our manuscript, including contents and figures.
- Magnani, E., Hake, S. KNOX lost the OX: the Arabidopsis KNATM gene defines a novel class of KNOX transcriptional regulators missing the homeodomain. Plant Cell 2008, 20, 875-887, doi: 10.1105/tpc.108.058495.
33. Hamant, O., Pautot V. Plant development: A TALE story. C. R. Biologies 2010, 333, 371-381, doi: doi.org/10.1016/j.crvi.2010.01.015
Reviewer 3 Report
The authors describe the TALE gene family in Punica granatum. This analysis is re-using publicly available data sets for the characterisation of this gene family on the sequence level and also to investigate the transcriptional activity of genes. Standard methods are used to perform these analysis. Overall, the study is very descriptive without any mechanistic insights. Most sections of the manuscript are clearly written in standard English, but some parts need attention (see list below). The authors might want to investigate selected aspects in more detail (see suggestions below). Some presented conclusions lack the necessary evidence and the authors should generally describe the fidelity and limitations of their analyses/findings.
1) line73-line76: Currently, it is not clear which TALE sequences have been used as baits in this analysis. The TALE sequences retrieved from the specified databases should be included in a multiple FASTA file in the supplements. In addition, the IDs of these sequences need to be given.
2) The authors downloaded 'genome data' and performed a BLASTp. It is not clear which annotation version was used for this analysis. In addition, the authors need to explain why BLASTp and not HMMER was used for the identification of candidates. Using an e-value cutoff for the identification is not the best approach. The authors should build a phylogenetic tree with all possible candidates and decide about TALE family membership based on this tree. What is about genes that are not included in the annotation? The authors should consider approaches like tBLASTn to find such genes in the genome sequence.
3) line86: Incomplete sequences can still be bona fide members of a gene family. These pseudogene candidates should be included in the analyses to understand their loss of function.
4) line93-98: The authors should try and evaluate different tools for the construction of the phylogenetic tree. MAFFT for the alignment and RAxML for the construction of a phylogenetic tree would be a good alternatives. Was there partitioning used for the different domains in the TALEs? How was the phylogenetic tree 'optimized'?
5) line103: This perl script should be made available. Uploading to an established repository like github would be the best solution. Inclusion in the supplements would be an alternative.
6) Parameter and a proper reference are required for PlantCARE.
7) There are currently 125 RNAseq data sets of Punica granatum at the Sequence Read Archive. The authors should include all of them to investigate the expression of these TALEs in a broad range of tissue and conditions. The list of samples could be moved to the supplements. Comparison of different sequencing technologies is tricky though. The authors should comments on potential limitations when comparing Illumina and 454 data sets. A co-expression analysis might be useful to predict potential target genes.
8) It seems that BLASTp was not relevant as all genes were already detected by HMMER. The authors might want to check this and write the manuscript more concisely.
9) The properties of the encoded peptides of the candidate genes were predicted not analyzed. The authors need to clearly distinguish between genes and proteins in their manuscript. It is very important to clearly state that only predictions were made here. line151 generates the impression that the TALE localisation was actually studied. The authors need to rephrase that sentence.
10) line152-line154: Is it possible that this paragraph is part of the initial template and should be removed from the manuscript?
11) A new phylogenetic tree with all closely related species should be constructed to infer conclusion about duplications/deletions in comparison to these related species.
12) Finding similarity through reference-based modeling is not 'remarkable significant', but an inherent bias of the method. Such a model with always show a high similarity despite differences in the sequences. The authors should rephrase this paragraph.
13) Ramachandran plot is not an assessment of the model quality, but displays protein properties (line203). The authors might want to rephrase the sentence.
14) The authors need to express the certainty of their findings clearly. The function prediction does not "reveal" a function, but only "predicts" a function (line211). The authors might want to rephrase the entire paragraph.
15) The authors should analyse protein-protein interaction with respect to BELL-KNOX heterodimers. These are mentioned in the introduction and could be very interesting.
16) Tool names and technical details of the analysis should only be presented in the method section and not in the results (e.g. line227-line228).
17) Many cis-regulatory elements occur frequently in the genome sequence just by chance. The authors might want to integrate an analysis (e.g. a comparison to random sequences of the genome) to indicate the relevance of their findings. Are there actually more cis-regulatory elements in these promoters than elsewhere?)
18) line278-line290 should be moved to the introduction. This is general background about TALEs. The authors should focus on their own results in the discussion and put them into context with existing studies.
19) line291-line294: Similar protein properties are a consequence of overall sequence similarity. Therefore, this finding is trivial and should not be presented in detail. If there are interesting differences between the predicted protein properties in this species and previous published work, the authors could focus on these differences.
20) The authors presented different classification systems for the TALE gene family members. Why was the system with 6 groups selected in this study?
21) line298-line315: Additional analysis are required to allow conclusions about an enrichment of hormone-regulated elements.
22) Where is the evidence for "It has also been objectively confirmed that PgTALE has a particular role in maintaining flower organ and fruit development." (line325-line327)?
23) The authors might want to check which cis-regulatory element is missing in the PgTALE13 promoter compared to closely related species. Finding a mechanism responsible for the inactivity of this gene would be really interesting. What is the function of PgTALE13 and why is this function not required in P. granatum?
Minor comments:
- line19: PgTALE is not the name of one gene. A number might be missing here.
- line23-25: This sentence needs to be rephrased as a gene family and not a single gene was studied here.
- line31: 'folded in three dimensions' should be removed as all proteins are folded in 3D
- line50: rephrase the sentence to better explain 'it'
- line67: 'genomic' > genome sequence
- line73: genome > genome sequence; transcriptome > transcriptome sequence
- line90: performed > predicted
- line 115-116: "The possible cis-elements in the promoter region were found." this would be a sentence of the results section
-line 140: Arabidopsis > Arabidopsis thaliana (check this throughout the manuscript)
- line159: species names need to be written in italics (check this throughout the manuscript)
- line338: The authors might want to check if there are two spaces between "conclusions are"
- line336: structure of the subfamily > structure of all members of the subfamily
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a point-by-point response to each comments.
Please see the attachment.
Please let me know if you have any questions.
Sincerely,
Yuying Wang
Author Response File: 
Author Response.docx
Round 2
Reviewer 3 Report
The authors integrated many of my comments and improved their manuscript substantially. There are only a few minor points remaining, but I am unable to inspect the supplementary files.
1) Perfect. Where is this mentioned in the manuscript?
2/3) The sequence analysis is substantially improved. There are two remaining points:
a) Genes encoding an incomplete domain could be considered as TALE-like or pseudogenes though. Maybe it would be helpful to include these sequences in the phylogenetic tree. Are these sequences copies of one degenerated gene or are they derived from multiple different genes through independent duplication and degradation events?
b) By searching a protein data sets, the outcome is biased by the annotation quality. Therefore, it is highly recommended to screen the genome sequence for additional genes. tBLASTn would be one option, but there are also other tools.
4) ok
5) Unfortunately, I am unable to access the supplements. The file appears to be broken.
6) ok
7) ok
8) I do not see the additional values, but it is also not detrimental to have a second method.
9) This sentence needs to be rephrased again. The prediction was done by a tool and not by the subcellular localization. Additionally, there are many new cases where genes and proteins need to be distinguished (in the added text).
10) ok
11) ok
12) ok
13) ok
14) Protein function predicted that > Protein function prediction suggested that
15) ok
16) ok
17) Where can I see the results of these controls?
18) ok
19) ok
20) This should be presented in the manuscript.
21) ok
22) ok
23) ok
Minor comments:
Gene names need to be written in titalic (e.g. line228).
Results and discussion need to be clearly separated (e.g. line 227).
line176: RAxM > RAxML
Author Response
Dear reviewer,
The authors would like to thank you for your careful work and thoughtful suggestions that have helped improve this paper substantially. The following is a point-by-point response to each comment.
The main revisions in the paper and the response to reviewer 3 commentsare as following:
Point 1: Perfect. Where is this mentioned in the manuscript?
Response1: Thanks, we mentioned in line93-95‘and the TALE family protein sequences of 6 species (A. thaliana, E. grandis, P. trichocarpa, M. domestica, V. viniferaand S. lycopersicum) as baits to make a local BLASTP alignment’.
Point 2/3:The sequence analysis is substantially improved. There are two remaining points:
- a) Genes encoding an incomplete domain could be considered as TALE-like or pseudogenes though. Maybe it would be helpful to include these sequences in the phylogenetic tree. Are these sequences copies of one degenerated gene or are they derived from multiple different genes through independent duplication and degradation events?
- b) By searching a protein data sets, the outcome is biased by the annotation quality. Therefore, it is highly recommended to screen the genome sequence for additional genes. tBLASTn would be one option, but there are also other tools.
Response: a):These sequences are derived from alternative splicing. This is the original words in the references‘Alternative splicing generates at leastthree KNATM isoforms that define a new KNOX phylogeneticclass.’
- Magnani, E., Hake, S. KNOX lost the OX: the Arabidopsis KNATM gene defines a novel class of KNOX transcriptional regulators missing the homeodomain. Plant Cell 2008, 20, 875-887, doi: 10.1105/tpc.108.058495.
Response: b):Based on your suggestion, we used tBALSTn to screen the genome sequence for additional genes, the result was consistent with HMMER.
Point 4: ok
Response 4: Thanks
Point 5: Unfortunately, I am unable to access the supplements. The file appears to be broken.
Response 5: We are sorry, so we provided the supplements again.
Point 6: ok
Response 6: Thanks
Point 7: ok
Response7:Thanks
Point 8: I do not see the additional values, but it is also not detrimental to have a second method.
Response 8: Thanks, in the future analysis, we will not use the BLASTPmethod.
Point 9: This sentence needs to be rephrased again. The prediction was done by a tool and not by the subcellular localization. Additionally, there are many new cases where genes and proteins need to be distinguished (in the added text).
Response 9: We have modified as ‘Subcellular localization prediction suggested that all PgTALE proteins were distributed on the nucleus.’and distinguished genes and proteins.
Point 10: ok
Response 10: Thanks
Point 11: ok
Response 11: Thanks
Point 12: ok
Response 12:Thanks
Point 13: ok
Response13: Thanks
Point 14: Protein function predicted that > Protein function prediction suggested that
Response 14: We have modified ‘Protein function prediction suggested that’
Point 15: ok
Response 15: Thanks
Point 16: ok
Response 16: Thanks
Point 17: Where can I see the results of these controls?
Response 17: We provided in the supplements(Table S3. All cis-elements of 17PgTALE)
Point 18: ok
Response18: Thanks
Point 19: ok
Response 19: Thanks
Point 20: This should be presented in the manuscript.
Response 20: We presented in line167-170‘Based on the classification of A. thalianaTALE gene family (BELL and KNOX family), the pomegranate BELL proteins were classified into 5 subfamilies: BELL-Ⅰ (one member), BELL-Ⅱ (two), BELL-Ⅲ (one), BELL-Ⅳ (two) and BELL-Ⅴ (three), and KNOX proteins were classified into 3 subfamilies: KNOX-Ⅰ (five), KNOX-Ⅱ (two), KNOX-Ⅲ (five).’
Point 21: ok
Response 21: Thanks
Point 22: ok
Response 22: Thanks
Point 23: ok
Response 23: Thanks
Minor comments:
Gene names need to be written in italic (e.g. line228).
Response: We have modified as gene names in italic.
Results and discussion need to be clearly separated (e.g. line 227).
Response: Thanks, we have modified it.
line176: RAxM > RAxML
Response: Thanks, we revised as‘RAxML’.
We sparedno effort to improve the manuscript and made revisions in the manuscript. We appreciate for your warm work earnestly, and hope the revisions will meet with approval.
