Characterization of TaMYB Transcription Factor Genes Revealed Possible Early-Stage Selection for Heat Tolerance in Wheat

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in green text color in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Thank you!

Are all the cited references relevant to the research?

Must be improved/No

The references have been reviewed carefully and updated.

Is the research design appropriate?

Yes

Thank you!

Are the methods adequately described?

Must be improved/No

All the method sections have been carefully reviewed and revised.

Are the results clearly presented?

Must be improved/No

The results section has been carefully reviewed and revised

Are the conclusions supported by the results?

Must be improved/No

The conclusion section has been carefully reviewed and revised

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

·         In 2.2 there is repeat of 2 lines and links in this repeats are different, and both links seem wrong.

·         In 2.3.1 no information about number of biological replicates declared, so it's impossible to judge the reliability of the statistics presented below.

·         In 2.3.2 there is very incomplete describing of experiment design - 12 pots for genotype, two sets, 12 pots in climate chamber, the reader have to find out by himself where are here two sets and why; what does mean two treatment of control; The words "The pots were returned to glasshouse after each treatment and grown till physiological maturity" applies to all plants or only those that have retained the flag leaf? In any sense, 3 biological replicates look too short for statistic.

Response 1:

Thank you for the comments. We have removed the duplicated lines from the text in 2.2 “and gene duplication (Sequence identity and similarity program) followed by selection of abiotic stress responsive genes based on in-silico expression analysis (Wheat expression database) [7].”. We also added the webpage link for wheat expression database to ensure the link provided is correct as https://www.wheat-expression.com/ on line 133 of page 4.

In 2.3.1, we have mentioned the number of biological replicates in the manuscript as “three biological replicates” on line 139 of page 4.

In 2.3.2, two treatments mean one for control and other for heat stress. So to make it more clear we have rephrased it as “and two treatments (one as control and another as HS)” on line 153 of page 4.

"The pots were returned to glasshouse after each treatment and grown till physiological maturity" applies to only plants with flag leaf. Therefore, we have rephrased the sentence as “The pots containing plants from set B, one with flag leaf intact, were returned to glasshouse after each treatment and grown till physiological maturity” from line 164 to 166 on page 4.

We strongly feel that three biological replicates are enough to serve our purpose of the research and many researchers use three biological replicates. We could only afford three biological replicates at the time. Of course, more will be better, we will take this into consideration in our future research.

Comments 2: It would be desirable to explain by what criteria 409 genes out of 1896 were selected, and then 48 of them were studied in the work. Re-naming of genes between tables 2 and 3 make difficult to compare them.

There is also no explanation what do authors mean writing about 876 non-redundant putative homologous genes out of 1896. In my opinion, the work will be more clear if all this transitions from one number to another are clearly justified.

The phrase regarding DEGs of Yitpi in the penultimate paragraph of section 3.5.3 requires clarification "at 13DPA"

Response 2:

We agree. In this study, the 1896 putative homologs of MYB genes were selected based on the following criteria as described in the manuscript “The latest Hidden Markov Model (HMM) profile of the MYB and MYB-related domains (PF00249, PF13921, PF14215, PF11831, and PF14379) downloaded from the protein family (Pfam) database (http://pfam.xfam.org/family/PF00847) [35], were used as a query sequence to HMM search using HMMER3.1b2 software (http://hmmer.org/) in the wheat reference protein sequence version 1.1 (http://plants.ensembl.org/Triticum_aestivum/Info/Index), with an E-value threshold of E <1e-5.”  in section 2.1 from line 118 to 124 on page 3. Further scaling down in number of genes from 1896 to 409 was done based on different criteria as described in section 2.2 as “Putative MYB genes identified were grouped into different sub-families based on the NCBI-CD search (https://www.ncbi.nlm.nih.gov/cdd) and the number of genes were reduced using Treetrimmer principle” from line 126 to 128 on page 4.

The final 48 genes were selected based on the in-silico expression analysis in response to abiotic stress as mentioned in section 2.2 from line 132 to 133 on page 4.

Regarding the renaming of genes in Table S2 and Table S3, the same ‘Gene ID’ mentioned in Table S2 have been provided beside the ‘Gene Name’ on Table S3, therefore we considered it makes the tables clear enough to track the “Gene Name” for each “Gene ID”.

The 876 non-redundant putative homologous genes out of 1896 means there were some duplicated genes present in putative homologous genes derived from the five different PF families, we used as query sequence. Therefore, we removed the duplicates and considered only the non-duplicated/ non-redundant genes in further analysis.

In section 3.5.3, in Yitpi, only one DEG was expressed uniquely at both seedling and 11DPA, while three DEGs at 13DPA. To make it clear we have rephrased the part as “The number of uniquely expressed DEGs were one (TaMYB-161) at seedling stage, one (TaMYB-267) at 11DPA and three (TaMYB-352, TaMYB-273, and TaMYB-356) at 13DPA” on line 340 to 342 on page 9.

Comments 3: In 2.2 there are two wrong links and it will need to check all links for a case. It can also be useful to give some more fresh ones and describe some regulations ways of this TF.

Response 3:

We agree, in 2.2, the suggested revisions have been made and link for the database has also been provided. The remaining links provided in the manuscript has also been checked and are all correct.

Major comments

Comment 1: Clear explanations about choose of genes for the research, description and figure of their structure. If there is an information about genes up or down them in regulation chains it would make more sense to the research.

Response 1:

The explanations for choosing MYB genes for this research has been provided in the introduction section from line 71 to 97 on page 2 and 3. We have added the following sentence “A previous transcriptomic research conducted in our lab found that among 60 TF families, MYB and MYB-related TF were top TF families responsible for abiotic stress defence in wheat [34]” in the text from line 95 to 97 on page 3.

However, the genes up- or down-regulated in the gene regulation have not been studied in this manuscript, which could be a good work following the outputs of this research.

Comment 2: Quantity of biological replicates. Yes, the minimum for ANOVA is 3, but not in biology research, could be used just for PCR. 10 is minimum minimorum for morphological comparison, 5 - for biochemistry.

Response 2:

We have used three biological replicates in this research, which is based on our literature review where we found many researches used three biological replicates such as Lu et al 2022 (doi:10.1016/j.cj.2022.01.005), Al-Ashkar et al 2023 (doi:10.3390/agronomy13010154.), Hasan et al 2024 (https://doi.org/10.1016/j.heliyon.2024.e38623), Sallam et al 2024 (https://doi.org/10.3390/agronomy14091999), etc.

We strongly feel that three biological replicates are enough to serve our purpose of the research. We could only afford three biological replicates at the time. Of course, more will be better, we will take this into consideration in our future research.

Comment 3: In Discussion you have no basis to conclude that the DEGs identified are responsible for HS resistant or sensibility until this is confirmed experimentally by silencing or overexpression of this genes. The result of the work is the discovery of genes, associated with traits, the nature of this connection has not yet been determined. In this way it could be interesting also to discuss a bit genes, commonly expressed between resistant and sensitive genotypes.

Response 3:

We agree, the confirmation of the genes can be done experimentally using gene silencing or overexpression techniques. We have changed “responsible for” to “associated with” in the manuscript when relevant to avoid this issue from line 474 to 477 on page 2.

To address the genes commonly expressed between resistant and sensitive genotypes we have added “In addition, some DEGs were expressed commonly between the tolerant and sensitive genotypes, where one DEG (TaMYB-299) at seedling stage, six DEGs (TaMYB-352, TaMYB-273, TaMYB-296, TaMYB-377, TaMYB-363, and TaMYB-304) at 11DPA and 10 DEGs (TaMYB-288, TaMYB-287, TaMYB-286, TaMYB-292, TaMYB-375, TaMYB-053, TaMYB-161, TaMYB-369, TaMYB-140, and TaMYB-304) at 13DPA were commonly expressed between Perenjori and Yitpi. Similarly, three DEGs (TaMYB-261, TaMYB-138, and TaMYB-275) at seedling stage, four DEGs (TaMYB-287, TaMYB-375, TaMYB-399, and TaMYB-361) at 11DPA and three DEGs (TaMYB-014, TaMYB-206, and TaMYB-226) at 13DPA were commonly expressed between Perenjori and Brazil32 (Figure 4). These commonly expressed genes between tolerant and sensitive genotypes might be associated with other common developmental functions characteristic to the MYB gene family.” In conclusion section from line 514 to 524 on page 3.

Detail comments

Comment 1: Please, check the Introduction part, there are some phrase need to be rewrite, like 2nd and 3rd ones.

In " Heat induced expression of MYB TF genes has also been reported in several plant species; AtMYB68 and AtMYB30 in transgenic Arabidopsis [21,22]; Os-MYB55 and OsMYB1 in rice [23,24]; LiMYB305 in transgenic Arabidopsis [25]; LeAN2 in transgenic tomato [26]; BnaMYB111L in tobacco [27]; BdMYB056 and BdMYB091 in Brachypodium [28]." you need to combine two parts about Arabidopsis.

Response 1:

Agreed, the 2^nd and 3^rd sentence has been revised as “Heat stress significantly impacts on plant growth and development through damage in photosynthetic machinery, membrane permeability, protein dysfunction, ROS production and increased oxidative stress in plant cells [1,2]. At the same time, a 2°C increase in average temperature during wheat growing season is estimated to reduce grain production by 50% in Australia [3].” from line 33 to 37 on page 1.

The other sentence has been revised as suggested to combine two Arabidopsis parts as " Heat induced expression of MYB TF genes has also been reported in several plant species; AtMYB68, AtMYB30, and LiMYB305 in transgenic Arabidopsis [19,27,28]; Os-MYB55 and OsMYB1 in rice [29,30]; LeAN2 in transgenic tomato [31]; BnaMYB111L in tobacco [32]; and BdMYB056 and BdMYB091 in Brachypodium [33]” from line 83 to 87 on page 3.

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1:

The language has been thoroughly improved according to the reviewer’s comments.

5. Additional clarifications

None apart from the above.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in green text color in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Thank you!

Are all the cited references relevant to the research?

Yes

Thank you!

Is the research design appropriate?

Yes

Thank you!

Are the methods adequately described?

Yes

Thank you!

Are the results clearly presented?

Yes

Thank you!

Are the conclusions supported by the results?

Yes

Thank you!

3. Point-by-point response to Comments and Suggestions for Authors

Major comments

The manuscript entitled "Characterization of TaMYB Transcription Factor Genes Revealed Possible Early Stage Selection for Heat Tolerance in Wheat" has written well. Overall, this paper presents valuable insights into wheat heat tolerance and transcription factors but it can be improved in clarity, discussion depth, and methodological justification.

The following points need to be addressed to improve further quality of manuscript.

Comments 1: Consider mentioning the significance of this study in a broader context, such as its potential impact on wheat breeding programs.

Response 1:

This research provides a broader understanding on heat stress responsive expression pattern of MYB genes at different growth stages of wheat genotypes. The following sentences have been added into the text from line 535 to 538 on page 4 to address this. “The identified genes can be used for further investigation of target genes and proteins in regulatory network directly expressed due to HS. It provides an idea on how MYB may regulate HS tolerance in wheat, which helps in sustainable wheat breeding for heat tolerance.”          

Comments 2: The introduction effectively presents the problem of heat stress in wheat but could provide a stronger rationale for focusing on MYB transcription factors.

Response 2:

Agreed, we have, accordingly, revised to emphasize this point. “Also, previous root transcriptome profiling research conducted in our lab has reported that among 60 TF families, MYB and MYB-related TF were top TF families responsive to abiotic stress defense in wheat [39].” has been added in the text from line 95 to 97 on page 3.

Comments 3: Some citations lack recent references; consider including the latest research on heat tolerance mechanisms in wheat

Response 3:

Agreed, we have added recent references as “Heat stress significantly impacts plant growth and development by altering the phenology and physiology of plants through damage in photosynthetic machinery, membrane permeability, protein dysfunction, ROS production and increased oxidative stress in plant cells [1,2]” from line 33 to 35 on page 1.

To emphasize the role of MYB TF in heat tolerance mechanism we have added “MYB TFs play crucial role in heat tolerance mechanism by regulating the calcium signaling pathway, where absence of MYB TF increases the [Ca^2+]cyst under HS and enhance HS tolerance [19]. In addition, mechanism of MYB TF in regulating different biotic and abiotic stresses has broadly been discussed by [2,20].” from line 71 to 75 on page 2.

Comments 4: The methodology is detailed, but it would be useful to clarify the selection criteria for the 48 TaMYB genes analyzed via RT-qPCR.

Response 4:

The final 48 genes used in RT-qPCR analysis were selected based on the in-silico expression analysis in response to abiotic stress as mentioned in section 2.1 and 2.2 from line 118 to 133 on page 3 and 4 ,or more detailed response to this point, please see Response 2 to Reviewer 1.

Comment 5: The statistical analysis section could be more explicit in explaining how significance was determined for gene expression changes.

Response 5:  

The gene expression changes were compared between treatment and control for tolerant and sensitive genotypes and the Log₂FC values >=1 and <=-1 were considered significant, as mentioned from line 192 to 195 on page 5 in the manuscript.

Comment 6: The results are comprehensive, but it might help to include a more explicit comparison of heat tolerance mechanisms between tolerant and sensitive genotypes.

Response 6:

We have mainly focused on the genes expressed in tolerant and susceptible genotypes of wheat at seedling and reproductive stages. So, discussion about the comparison of heat tolerance mechanism between tolerant and sensitive genotypes is a byproduct.

Comments 7: Figures and tables are well-structured, but additional annotations in figures (e.g., highlighting key gene expression patterns) could improve readability.

Response 7:

Agreed, we have added “significantly upregulated/downregulated” in the legend of Figure 5 as shown on line 383 on page 5.  

Comments 8: The discussion successfully connects findings to previous studies, but it could expand on the implications for wheat breeding strategies.

Response 8:

Thanks, the identified genes can be used as the target genes for genomic selection. In addition, gene based markers can be developed based on our study, and used in marker-assisted selection in breeding.

Comments 9: Consider addressing potential limitations of using MYB genes as markers for early-stage selection.

Response 9:

The challenge is that many MYB genes have similar sequences which means that it may be difficult to develop gene-specific markers but it is possible. Therefore, genomic selection may be the better solution for breeding.

Comments 10: The correlation between seedling-stage expression and reproductive-stage performance is interesting; more emphasis on the biological significance of this correlation would strengthen the argument.

Response 10:

Agreed, we have added ‘morphologically heat tolerant seedlings of wheat produced higher yield at adult stage as compared to susceptible seedlings [5]” highlighting the biological significance of correlation between seedling and reproductive stage performance from line 41 to 43 on page 2.

Comments 11: The conclusion effectively summarizes key findings, but a brief mention of future research directions (e.g., validating these genes in field trials) would add value.

Response 11:

Agreed, it has been added accordingly. “These genes can also be further validated in field trials with multiple breeding lines to ensure the consistency in gene expression across lines.” has been added from line 538 to 540 on page 4.

Comments 12: The references are well-organized but should be updated with more recent literature where applicable.

Response 12:

Agreed, the references have been updated accordingly. Please also see Response 3 under the same section.

Minor issues:

Comments 1: Please correct "there-fore" and "in-creased"

Response 1:

Many thanks, they have been corrected accordingly.

Comments 2: "meristem injury and subsequent stem collapse in plants." Refine this sentence

Response 2:,

Thanks, it has been modified as “At seedling stage, HS affects seedling establishment due to higher seedling mortality [6], restricted nutrient absorption [7], disruption in leaf growth limiting photosynthetic area [8], and damage of vascular tissues resulting stunted growth and subsequent stem collapse in plants [9]“ from line 44 to 47 on page 2.

Comments 3: "phenology and physiology of plants through damage in photosynthetic machinery" rewrite the sentence

Response 3:

It has been rewritten as “Heat stress significantly impacts plant growth and development through damage in photosynthetic machinery, membrane permeability, protein dysfunction, ROS production and increased oxidative stress in plant cells [1,2].” from line 33 to 35 on page 1.

Comments 4: Ensure consistency in terms like "sub-families" vs. "subfamilies."

Response 4:

Many thanks, they have been corrected accordingly.

Detail comments

The manuscript entitled "Characterization of TaMYB Transcription Factor Genes Revealed Possible Early Stage Selection for Heat Tolerance in Wheat" has written well. Overall, this paper presents valuable insights into wheat heat tolerance and transcription factors but could benefit from minor refinements in clarity, discussion depth, and methodological justification.

The following points need to be addressed to improve further quality of manuscript.

Comment 1: The abstract provides a clear overview of the study but could benefit from more explicit details on the methodology used for identifying candidate genes.

Response 1:

Agreed. The following revisions have been made in the abstract by adding “and 48 genes were selected for qRT-PCR expression analysis based on in-silico expression analysis under abiotic stresses. “ from line 17 to 19 on page 1.

Comment 1: Consider mentioning the significance of this study in a broader context, such as its potential impact on wheat breeding programs.

Response:

This research provides a broader understanding on heat stress responsive expression pattern of MYB genes at different growth stages of wheat genotypes. The following sentences have been added into the text from line 533 to 538 on page 4.                         “The identified genes can be used for further investigation of target genes and proteins in regulatory network directly expressed due to HS. The research provides concepts on how MYB may regulate HS tolerance in wheat, which helps in sustainable wheat breeding for heat tolerance”.

Comment 2: The introduction effectively presents the problem of heat stress in wheat but could provide a stronger rationale for focusing on MYB transcription factors.

Response 2:

Agreed, we have, accordingly, revised to emphasize this point. “Also previous root transcriptome profiling research conducted in our lab has reported that among 60 TF families, MYB and MYB-related TF were top TF families responsive to abiotic stress defense in wheat [39].” has been added in the manuscript from line 95 to 97 on page 3.

Comment 3: Some citations lack recent references; consider including the latest research on heat tolerance mechanisms in wheat

Response 3:

Agreed, we have added recent references as “Heat stress significantly impacts plant growth and development by altering the phenology and physiology of plants through damage in photosynthetic machinery, membrane permeability, protein dysfunction, ROS production and increased oxidative stress in plant cells [1,2]” from line 33 to 35 on page 1.

To emphasize the role of MYB TF in heat tolerance mechanism we have added “MYB TFs play crucial role in heat tolerance mechanism by regulating the calcium signaling pathway, where absence of MYB TF increases the [Ca^2+]cyst under HS and enhance HS tolerance [19]. In addition, mechanism of MYB TF in regulating different biotic and abiotic stresses has broadly been discussed by [2,20].” from line 71 to 75 on page 2.

Comment 4: The methodology is detailed, but it would be useful to clarify the selection criteria for the 48 TaMYB genes analyzed via RT-qPCR.

Response 4:

The final 48 genes used in RT-qPCR analysis were selected based on the in-silico expression analysis in response to abiotic stress as mentioned in section 2.1 and 2.2 from line 118 to 133 on page 3 and 4 ,or more detailed response to this point, please see Response 2 to Reviewer 1.

Comment 5: The statistical analysis section could be more explicit in explaining how significance was determined for gene expression changes.

Response 5:  

The gene expression changes were compared between treatment and control for tolerant and sensitive genotypes and the Log₂FC values ≥1 and ≤-1 were considered significant, as mentioned from line 192 to 195 on page 5 in the manuscript.

Comment 6: The results are comprehensive, but it might help to include a more explicit comparison of heat tolerance mechanisms between tolerant and sensitive genotypes.

Response 6:

We have mainly focused on the genes expressed in tolerant and susceptible genotypes of wheat at seedling and reproductive stages. So, discussion about the comparison of heat tolerance mechanism between tolerant and sensitive genotypes is a byproduct.

Comment 7: Figures and tables are well-structured, but additional annotations in figures (e.g., highlighting key gene expression patterns) could improve readability.

Response 7:

Agreed, we have added “significantly upregulated/downregulated” in the legend of Figure 5 as shown on line 383 of page 5. 

Comment 8: The discussion successfully connects findings to previous studies, but it could expand on the implications for wheat breeding strategies.

Response 8:

Thanks, the identified genes can be used as the target genes for genomic selection. In addition, gene based markers can be developed based on our study, and used in marker-assisted selection in breeding.

Comment 9: Consider addressing potential limitations of using MYB genes as markers for early-stage selection.

Response 9:

The challenge is that many MYB genes have similar sequences which means that it may be difficult to develop gene-specific markers but it is possible. Therefore, genomic selection may be the better solution for breeding.

Comment 10: The correlation between seedling-stage expression and reproductive-stage performance is interesting; more emphasis on the biological significance of this correlation would strengthen the argument.

Response 10:

Agreed, we have added ‘morphologically heat tolerant seedlings of wheat also produced higher yield at adult stage as compared to susceptible seedlings [5]” highlighting the biological significance of correlation between seedling and reproductive stage performance from line 41 to 43 on page 2.

Comment 11: The conclusion effectively summarizes key findings, but a brief mention of future research directions (e.g., validating these genes in field trials) would add value.

Response 11:

Agreed, it has been added accordingly. “These genes can also be further validated in field trials with multiple breeding lines to ensure the consistency in gene expression across lines.” has been added from line 538 to 540 on page 4.

Comment 12: The references are well-organized but should be updated with more recent literature where applicable.

Response 12:

Agreed, the references have been updated accordingly. Please also see Response 3 under the same section.

Comment 13: Please provide the figures of phenotype, if possible

Response 13:

Figure for phenotype at seedling stage has been provided in supplementary Fig. 3 in the manuscript and for reproductive stage glasshouse experiment has been added as citation “at reproductive stage (Supplementary Figure 4) from line 256 to 257 on page 7 and enlisted with legend “Glass house experiments showing the three different stages of wheat growth (a) sowing, (b) vegetative growth, and (c) reproductive growth approaching physiological maturity.” from line 583 to 585 on page 5.

Minor issues:

Issue 1: Please correct "there-fore" and "in-creased"

Response 1:

Many thanks, they have been corrected accordingly.

Issue 2: "meristem injury and subsequent stem collapse in plants." Refine this sentence

Response 2:

Thanks, it has been modified as “At seedling stage, HS affects seedling establishment due to higher seedling mortality [6], restricted nutrient absorption [7], disruption in leaf growth limiting photosynthetic area [8], and damage of vascular tissues resulting stunted growth and subsequent stem collapse in plants [9]“ from line 44 to 47 on page 2.

Issue 3: "phenology and physiology of plants through damage in photosynthetic machinery" rewrite the sentence

Response 3:

It has been rewritten as “Heat stress significantly impacts plant growth and development through damage in photosynthetic machinery, membrane permeability, protein dysfunction, ROS production and increased oxidative stress in plant cells [1,2].” from line 33 to 35 on page 1.

Issue 4: Ensure consistency in terms like "sub-families" vs. "subfamilies."

Response 4:

Many thanks, they have been corrected accordingly.

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1:

The language has been thoroughly improved according to the reviewer’s comments.

5. Additional clarifications

None apart from the above.