Comprehensive Analysis of ZmTBL Genes Reveals Their Roles in Maize Development and Abiotic Stress Responses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted article is dedicated to a completely new study - a timely study of the TBL family genes in Zea mays. The authors performed extensive genomic screening, bioinformatic annotation, expression analysis under abiotic stress, and association analysis of SNP markers with agronomic traits.

The presented work attracts the reader's interest. Despite the high level of the analysis performed, the manuscript has a number of shortcomings that require attention.

1. Identification and functional analyses of ZmTBL genes reveal their roles involved in maize development and abiotic stress responses – It's better not to use the word "involved" here, but simply write «… their roles in maize development and abiotic…»
2. There is a technical error in the keywords – “Association analyse” should be corrected as “Association analysis”. I suggest that in order to avoid repetitions with the title in the keywords, write “Zea mays” instead of “Maize”
3. In Introduction-It would be more correct to write TBL proteins on lines 50 and 61, because those that perform the direct function are already proteins.
4. In Introduction - (Zea mays) written on line 84 – should be given on line 75, where maize is first mentioned.
5. In the methods section, the software used to design the primers should be indicated.
6. In the Results section – In the text, Fig. 1 refers to the phylogenetic analysis of proteins, but the figure caption says “Phylogenetic analysis of TBL genes of different species.”
7. In general, it is confusing for the reader to distinguish between your own results and the expression results you analyze bioinformatically. To distinguish the former from the laboratory analysis, it would be more appropriate to call the former an in silico expression analysis.
8. Comparative analyses were poorly conducted in the Discussion section. The authors presented their own results more. A more in-depth discussion with modern statistical analysis would have increased the value of the article.

Author Response

Comments 1: Identification and functional analyses of ZmTBL genes reveal their roles involved in maize development and abiotic stress responses – It's better not to use the word "involved" here, but simply write «… their roles in maize development and abiotic…»

Response 1: Thank you for your pointing this out. We agree with this comment. Therefore, we have revised our title, which can be found on line 3, page 1 in our revised manuscript.

Comments 2: There is a technical error in the keywords – “Association analyse” should be corrected as “Association analysis”. I suggest that in order to avoid repetitions with the title in the keywords, write “Zea mays” instead of “Maize”

Response 2: Thank you for your pointing this out. We agree with this comment. Therefore, we have revised our keywords according to your advice. Please see our revision on line 30, page 1 in our revised manuscript.

Comments 3: In Introduction-It would be more correct to write TBL proteins on lines 50 and 61, because those that perform the direct function are already proteins.

Response 3: We would like to thank the reviewer for his/her careful review, we have replaced “TBL genes” with “TBL proteins” on lines 50 and 65 in our revised manuscript.

Comments 4: In Introduction - (Zea mays) written on line 84 – should be given on line 75, where maize is first mentioned.

Response 4: We would like to thank the reviewer for his/her careful review, we have given (Zea mays) on line 85 instead of line 94 in our revised manuscript.

Comments 5: In the methods section, the software used to design the primers should be indicated.

Response 5: Thank you for your pointing this out. In our study, we designed the primers by using Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and we add this information in methods, please see our revision on line 162.

Comments 6: In the Results section – In the text, Fig. 1 refers to the phylogenetic analysis of proteins, but the figure caption says “Phylogenetic analysis of TBL genes of different species.

Response 6: Thank you for your pointing this out, we have revised the description in the figure caption, please see our revision on line 190, page 5 in our revised manuscript.

Comments 7: In general, it is confusing for the reader to distinguish between your own results and the expression results you analyze bioinformatically. To distinguish the former from the laboratory analysis, it would be more appropriate to call the former an in silico expression analysis

Response 7: Thank you for your pointing this out. We agree with this comment. To make a better distinction between expression results analyzed bioinformatically and laboratory analysis, we have revised the presentation in our manuscript, and expression results analyzed bioinformatically were designated in revised manuscript.

Comments 8: Comparative analyses were poorly conducted in the Discussion section. The authors presented their own results more. A more in-depth discussion with modern statistical analysis would have increased the value of the article.

Response 8: We would like to thank the reviewer for his/her valuable suggestions. Therefore, we had a more in-depth discussion of our research content in discussion section. Please see the discussion section our revision manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors made a deep bioinformatics analysis and qPCR expression profiling of TBL genes in maize

only few comments:

sometimes is used italics to refer a genes name, but others don´t

pag 2, lines 50 ,51, 74, 86 TBL genes 

lines 93, 103, 138 ZmTBL

pag 3 line 63s 114, 115 Oryza sativa,  A. thaliana

essential for leaf blight resistance in rice (why is writing with uppercase the first letter?)

Line 83 and 84, D. officinale, Zea mays

Author Response

Comments 1: sometimes is used italics to refer a genes name, but others don´t

pag 2, lines 50 ,51, 74, 86 TBL genes 

lines 93, 103, 138 ZmTBL

pag 3 line 63s 114, 115 Oryza sativa,  A. thaliana

essential for leaf blight resistance in rice (why is writing with uppercase the first letter?)

Line 83 and 84, D. officinale, Zea mays

Response 1: We would like to thank the reviewer for his/her carefully reviewing the content and format of our manuscript. We have made careful modifications to the italicized format of genes and species names in the article, which can be observed in the revised manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Peer review:

Title: "Identification and functional analyses of ZmTBL genes reveal their roles involved in maize development and abiotic stress responses"

General Overview:

This study presents a comprehensive analysis of the Trichome Birefringence-Like (TBL) gene family in maize (Zea mays), which encodes polysaccharide O-acetyltransferases involved in cell wall modification. It provides valuable insights into the functional diversity and breeding potential of the ZmTBL gene family in maize. However, there are some concerns that need to be addressed. They are as follows:

1. Wrong spellings:

P2, L91; ‘growth condition’ should be ‘growth conditions’.

P4, L156; ‘The MLM model were’ should be ‘The MLM model was’.

P4, L167; ‘ZmTBLs belongs to group I/IV/V/VI’ should be ‘ZmTBLs belonging to group I/IV/V/VI’. It applies to all similar mistakes.

P9, L256; ‘analyse between’ should be ‘analysis between’.

P12, L349; ‘indicates that TBL protein’ should be ‘indicating that TBL protein’.

P13, L395; ‘Natural variations are basis..’ should be ‘Natural variations are the basis..’.

P13, L401; ‘through association analyse’ should be ‘through the association analysis’.

P14, L418; ‘environmental stresse’ should be ‘environmental stresses’.

In all, the author should get professional help to correct all the grammatical mistakes in this manuscript.

2. The title is too long; it is redundant, and the author should simplify it to improve the impact and readability.
3. The abstract lacks quantitative highlights. The maize TBL genes lack functional verification. No clear genetic evidence is shown to demonstrate that ZmTBL57 and ZmTBL69 are related to maize agronomic traits. This manuscript stops at the early-stage predictions, which makes its scientific significance relatively low.
4. The author should add brief summarizing statements at the end of each major subsection to integrate findings.
5. The Discussion section repeats many of the results without deeper interpretation.
6. The conclusion mostly restates results without offering clear take-home messages or future directions.

In summary, while the manuscript provides a genome-wide analysis of the ZmTBL gene family in maize, the findings are largely descriptive and lack novel mechanistic insights. Substantial improvement in experimental validation and clearer functional interpretation are needed to enhance its contribution to the field.

 

 

Comments on the Quality of English Language

It must be improved.

Author Response

Comments 1: Wrong spellings:P2, L91; ‘growth condition’ should be ‘growth conditions’.

P4, L156; ‘The MLM model were’ should be ‘The MLM model was’.

P4, L167; ‘ZmTBLs belongs to group I/IV/V/VI’ should be ‘ZmTBLs belonging to group I/IV/V/VI’. It applies to all similar mistakes.

P9, L256; ‘analyse between’ should be ‘analysis between’.

P12, L349; ‘indicates that TBL protein’ should be ‘indicating that TBL protein’.

P13, L395; ‘Natural variations are basis..’ should be ‘Natural variations are the basis..’.

P13, L401; ‘through association analyse’ should be ‘through the association analysis’.

P14, L418; ‘environmental stresse’ should be ‘environmental stresses’.

In all, the author should get professional help to correct all the grammatical mistakes in this manuscript.

Response 1: We would like to thank the reviewer for his/her carefully reviewing the content and format of our manuscript. We have carefully corrected the wrong spellings and grammar mistakes in our manuscript, which can be observed in the revised manuscript.

Comments 2: The title is too long; it is redundant, and the author should simplify it to improve the impact and readability.

Response 2: Thank you for your pointing this out. According to your advice, we have simplify our title to improve the readability.

Comments 3: The abstract lacks quantitative highlights. The maize TBL genes lack functional verification. No clear genetic evidence is shown to demonstrate that ZmTBL57 and ZmTBL69 are related to maize agronomic traits. This manuscript stops at the early-stage predictions, which makes its scientific significance relatively low.

Response 3: Thank you for your pointing this out. We also acknowledge the lack of functional validation of ZmTBLs in our study, which represents a limitation of the current research. Indeed, to get a better insight of ZmTBLs’ functions, further experimental validations are needed. And we will experimentally validate the specific functions of ZmTBLs in our future study. As no ZmTBL gene has been functional validated up to now, we believe our study will contribute to future functional research on ZmTBL genes, and we also discussed this in the discussion section (line 441-442) of our revised manuscript.

Comments 4: The author should add brief summarizing statements at the end of each major subsection to integrate findings.

Response 4: Thank you for your pointing this out. According to your advice, we have added brief summarizing statements at the end of each major subsection to integrate findings. Please see the result section of our revised manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

In this study, the authors claimed that they had identified 74 maize TBL genes (designated ZmTBL1–ZmTBL74) among maize genomes. According to their studies, comprehensive analyses of their phylogenetic relationships, gene structures, chromosomal distributions, putative upstream regulatory transcription factors, and expression patterns were also conducted. Their results revealed that ZmTBLs respond widely to abiotic stresses, including heat and cold. Moreover, association analyses demonstrated that sequence variations in ZmTBL57 and ZmTBL69 correlate with maize agronomic traits.

Overall., the study is interesting and informative, but it lacks some important points which should be addressed.

1. What is basis of selection of TBL genes? please provide factual information.
2. What is the mechanistic action of TBL genes in plant growth and development? Please provide factual information in introduction section.
3. In line 61 and 62 authors discussed Besides regulating growth and development, TBL genes also participate in various plant biotic and abiotic stress responses? How they play role as stress alleviator? Discuss in detail and also discuss to difference in mode of action of these genes in biotic and abiotic stresses alleviation with facts and figures.
4. Introduction section should be revised properly with factual information supporting the role of mechanistic action of mentioned genes.
5. In material and method line 96 to 103 I don’t see any references? please add references
6. Please add the gene, protein interaction and protein interaction in your study using STRING software and submit these gene sequences in any repository and add its link in MS before its publication
7. Provide functional categorization of genes sequences in details
8. Provide a figure showing functional categorization of genes and proteins.
9. Discussion section should be detailed. Provide recent references supporting your study
10. I am not satisfied with conclusion section please elaborate it keeping in mind future perspectives of your research study
11. References should be double check in terms of over citations and under citations
12. English grammar should be double checked and try to remove errors
13. Add company, model number of all instruments used for this study.

Author Response

Commonts 1: What is basis of selection of TBL genes? please provide factual information.

Response1: Thank you for your pointing this out. According to the identification results of Arabidopsis TBL members in previous study, we constructed a Hidden Markov Model (HMM) for identifying ZmTBL members by using HMMER software. Furthermore, based on the analysis of TBL member protein sequences identified in other species such as Arabidopsis, rice, cotton, and poplar, the PF13839 and PF14416 domains were found to be conserved domains widely present in TBL proteins. Consequently, we filtered out sequences from the HMMsearch results that lacked both domains. These procedures are described in section 2.2 of the materials and methods.

Comments 2: What is the mechanistic action of TBL genes in plant growth and development? Please provide factual information in introduction section.

Response2: Thank you for your pointing this out. TBL proteins are reported function as polysaccharide O-acetyltransferases that catalyze the O-acetylation of cell wall polysaccharides. The O-acetylation of polysaccharides are important to cell wall assembly and cell wall physico-chemical properties. For example, loss fuction of some TBL proteins (ESK1/TBL29, TBL34 and TBL35) can cause aberrant secondary wall structure due to xylan O-acetylation deficiency. This structural defect weakens cell wall strength, causing xylem vessel collapse, which subsequently impairs plant growth. Besides, pectin acetylation is critical for the gelling property of pectins and the elongation of cells, mutant of TBL44/PMR5 is defective in pectin acetylation and is also shown reduced cell expansion. We have provided these information in introduction section (line 57 to line 62) of our revised manuscript.

References:

1. Yuan Y, Teng Q, Zhong R, Ye ZH. Roles of Arabidopsis TBL34 and TBL35 in xylan acetylation and plant growth. Plant Sci. 2016, 243, 120-30.doi:10.1016/j.plantsci.2015.12.007.
2. Zhong R, Zhou D, Chen L, Rose JP, Wang BC, Ye ZH. Plant Cell Wall Polysaccharide O-Acetyltransferases. Plants (Basel). 2024, 13(16), doi: 10.3390/plants13162304.
3. Chiniquy D, Underwood W, Corwin J, Ryan A, Szemenyei H, Lim CC, Stonebloom SH, Birdseye DS, Vogel J, Kliebenstein D, Scheller HV, Somerville S. PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens. Plant J. 2019, 100(5), 1022-1035. doi: 10.1111/tpj.14497.

Comments 3: In line 61 and 62 authors discussed Besides regulating growth and development, TBL genes also participate in various plant biotic and abiotic stress responses? How they play role as stress alleviator? Discuss in detail and also discuss to difference in mode of action of these genes in biotic and abiotic stresses alleviation with facts and figures.

Response 3: Thank you for your pointing this out. In fact, many studies have shown that TBL genes participate in various plant biotic and abiotic stress responses. In cotton and rose, expression of TBL genes are regulated by various plant biotic and abiotic stresses such as cold, heat, drought and gray mold disease. Besides, the functions of many TBL genes in plant biotic and abiotic stresses have also been verified. For example, the TBL29/ESK1 in Arabidopsis plays a major role in plant response to water shortage and in whole plant water economy; TBR regulates plant Zn tolerance through modulating root cell wall pectin methylesterification, tbr mutant is hypersensitive to excess Zn and Zn-enhanced photomorphogenesis; OsTBL1 and OsTBL2, are essential for leaf blight resistance in rice by mediating Xylan Acetylation, ostbl1 single mutant and the tbl1 tbl2 double mutant displayed susceptibility to rice blight disease.

As cell wall is considered a protective barrier of plant cell, adjustment of cell wall  under stresses is an important phenomenon in plant adaptation. O-acetylation of polysaccharides are important to cell wall assembly and cell wall physico-chemical properties. Although it is widely accepted that function of TBL is achieved by O-acetylation of polysaccharides, mechanisms of TBL genes in regulating plant biotic and abiotic stresses remain to be further analyzed. We have supplemented relevant content in introduction section (line 67 to line 83) of our revised manuscript.

References:

1. Zhu X, Ma X, Hu W, Xing Y, Huang S, Chen Z, Fang L. Genome-wide identification of TBL gene family and functional analysis of GhTBL84 under cold stress in cotton. Front Plant Sci. 2024, 15, doi: 10.3389/fpls.2024.1431835.
2. Tian Y, Zhang S, Liu X, Zhang Z. Global Investigation of TBL Gene Family in Rose (Rosa chinensis) Unveils RcTBL16 Is a Susceptibility Gene in Gray Mold Resistance. Front Plant Sci. 2021, 12, doi: 10.3389/fpls.2021.738880.
3. Bouchabke-Coussa O, Quashie ML, Seoane-Redondo J, Fortabat MN, Gery C, Yu A, Linderme D, Trouverie J, Granier F, Téoulé E, Durand-Tardif M. ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance. BMC Plant Biol. 2008, 8, doi: 10.1186/1471-2229-8-125.
4. Zhong K, Zhang P, Wei X, Platre MP, He W, Zhang L, Małolepszy A, Cao M, Hu S, Tang S, Li B, Hu P, Busch W. Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification. Nat Commun. 2024, 15(1), doi: 10.1038/s41467-024-50106-5.
5. Gao Y, He C, Zhang D, Liu X, Xu Z, Tian Y, Liu XH, Zang S, Pauly M, Zhou Y, Zhang B. Two Trichome Birefringence-Like Proteins Mediate Xylan Acetylation, Which Is Essential for Leaf Blight Resistance in Rice. Plant Physiol. 2017, 173(1), 470-481. doi: 10.1104/pp.16.01618.

Comments 4: Introduction section should be revised properly with factual information supporting the role of mechanistic action of mentioned genes.

Response 4: Thank you for your pointing this out. We have supplemented relevant content in introduction section of our revised manuscript.

Comments 5: In material and method line 96 to 103 I don’t see any references? please add references

Response 5: Thank you for your pointing this out. We have added the corresponded reference in material and method.

Comments 6: Please add the gene, protein interaction and protein interaction in your study using STRING software and submit these gene sequences in any repository and add its link in MS before its publication.

Response 6: Thank you for your pointing this out. The expression correlation between ZmTBLs and potential upstream transcription factors was calculated using maize gene expression altas retrieved from the qTeller (https://qteller.maizegdb.org) within MaizeGDB. Our protein-protein interaction data was collected from MaizeNetome (http://minteractome.ncpgr.cn/) based on yeast two-hybrid method RLL-Y2H across the development of the maize inbred line B73, and we have supplemented these information in supplementary table S3 and S4.

References: 

1. Stelpflug SC, Sekhon RS, Vaillancourt B, Hirsch CN, Buell CR, de Leon N, Kaeppler SM. An Expanded Maize Gene Expression Atlas based on RNA Sequencing and its Use to Explore Root Development. Plant Genome. 2016,9(1). doi: 10.3835/plantgenome2015.04.0025.
2. Feng JW, Han L, Liu H, Xie WZ, Liu H, Li L, Chen LL. MaizeNetome: A multi-omics network database for functional genomics in maize. Mol Plant. 2023, 16(8), 1229-1231. doi: 10.1016/j.molp.2023.08.002.
3. Han L, Zhong W, Qian J, Jin M, Tian P, Zhu W, Zhang H, Sun Y, Feng JW, Liu X, Chen G, Farid B, Li R, Xiong Z, Tian Z, Li J, Luo Z, Du D, Chen S, Jin Q, Li J, Li Z, Liang Y, Jin X, Peng Y, Zheng C, Ye X, Yin Y, Chen H, Li W, Chen LL, Li Q, Yan J, Yang F, Li L. A multi-omics integrative network map of maize. Nat Genet. 2023 , 55(1), 144-153. doi: 10.1038/s41588-022-01262-1.

Comments 7: Provide functional categorization of genes sequences in details

Response 7: Thank you for your pointing this out, we have supplied the functional categorization of genes in Table S4.

Comments 8: Provide a figure showing functional categorization of genes and proteins.

Response 8: Thank you for your pointing this out, we have supplied the figure (see figure S5) to show functional categorization of genes and proteins.

Comments 9: Discussion section should be detailed. Provide recent references supporting your study

Response 9: Thank you for your pointing this out, we have detailed our conclusion section and provide recent references in our revised manuscript.

Comments 10: I am not satisfied with conclusion section please elaborate it keeping in mind future perspectives of your research study

Response 10: Thank you for your pointing this out, we have revised conclusion section in our manuscript.

Comments 11: References should be double check in terms of over citations and under citations

Response 11: Thank you for your pointing this out, we have rechecked and revised citations in our manuscript.

Comments 12: English grammar should be double checked and try to remove errors

Response 12: Thank you for your pointing this out, we have rechecked and revised grammar in our manuscript.

Comments 13: Add company, model number of all instruments used for this study.

Response 13: Thank you for your pointing this out, and we have provided the company and model number of all instruments used for this study in our methods part.