Identification and Expression Analysis of Wheat Golden2-like (TaGLK) Gene in Response to Biotic and Abiotic Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors' discovery of over 120 GLK genes/TF for wheat within the genome of its chromosome. However, the manuscript primarily focuses on bioinformatics data rather than laboratory experiments. That is the drawback of this study. I trust that the authors will also concur with this observation. The prediction of TMD, isoelectric point, and other basic bioinformatics analyses is often overlooked by researchers. I don't intend to discourage your efforts; however, conducting laboratory experiments is a more valid approach than generating more bioinformatics data. Besides, the authors only used a single tissue (leaves) for gene expression analysis. That is also not sufficient for gene expression analysis. Despite submitting their manuscript to the journal of agronomy, the authors failed to include any data on physiological, biomass, yield, or other agronomic traits. That is really disappointing. Therefore, the inclusion of several agronomic and biochemical traits by the authors significantly strengthens the manuscript. I don’t have any interest in rejecting the manuscript. I advised authors to try to include some agronomic data and clarify my doubts mentioned below.

1)      The authors should change the tile because they did not characterize the genes. They simply analysed gene expression analysis.

2)      In the abstract, authors should include the original results instead of bioinformatics analysis results.

3)      Is GLK Genes or TF? Kindly clarify and use the same pattern throughout the manuscript.

4)      At position 60, which disease resistance genes does GLK TF induce?

5)      On page 77, the authors noted that C-Repeat Binding Factor (CBF) transcription factors stimulate GLK genes. However, in L. No. 60 and other sections of the manuscript, the authors mention that GLK transcription factors induce disease resistance genes. I'm really confused. The authors should first clarify whether GLK is a gene or a transcription factor.

6)      Indeed, researchers have implicated several other candidate genes in enhancing biotic and abiotic stress tolerance in wheat, in addition to GLK genes/TF. Therefore, the authors should elucidate what distinguishes GLK genes from other genes. Why is it necessary to focus on GLK genes? It's crucial to emphasize this in the introduction section, as it elevates your study above existing reports.

7)      Sentences are not necessary for Nos. 96-97, as the authors collected the sequences directly from phytozome databases.

8)      The authors should specify in Section 2.1 which version of the wheat genome they used to mine genes from the phytozome database.

9)      How did the authors provide the gene names for Wheat GLK?

10)  Authors used only 40 GLK of rice and 10 GLK of Arabidopsis protein sequences to retrieve GLK genes for wheat. However, both plants do not possess more than 40 GLK proteins. Then how did authors collect the remaining GLK gene sequences for wheat? Authors should clarify and include these details in the materials and methods section.

11)  No. 110: Phytozome provides the chromosome number of any given gene. Why did the authors use the GFF3 file to determine the chromosomal location of the GLK gene? Kindly clarify this. What did you mean by the GFF file in this context?

12)  The authors should alter the title of Section 2.6, as those experiments are solely focused on bioinformatics.

13)  In Section 2.6, how does PEG600 differ from drought? The study also explored the impact of PEG600-induced drought stress. What is the selected stress basis for this experiment?

14)  NO. 165l; Jingshuang 16 cultivar resistance What abitoic tress? drought or cold or heat?

15)  The phylogenetic analysis revealed a close relationship between the majority of TaGLK proteins. How did the authors claim his? So, authors should reconstruct the phylogentic tree whose plant contains around 125 GLK proteins.

16)  Figure 4 displays only 99 GLK. Where are the results for the remaining 26 proteins?

17)  Which are the foundational genes chosen for the gene expression analysis experiment?

18)  The authors should include the temperature and product size of the primers used for gene expression analysis.

19)  Authors should include the position or location of each gene in the supplemental file, which will help identify whether authors found different genes or the same locus. This should be more important.

 

Author Response

Reviewer #1

I appreciate the authors' discovery of over 120 GLK genes/TF for wheat within the genome of its chromosome. However, the manuscript primarily focuses on bioinformatics data rather than laboratory experiments. That is the drawback of this study. I trust that the authors will also concur with this observation. The prediction of TMD, isoelectric point, and other basic bioinformatics analyses is often overlooked by researchers. I don't intend to discourage your efforts; however, conducting laboratory experiments is a more valid approach than generating more bioinformatics data. Besides, the authors only used a single tissue (leaves) for gene expression analysis. That is also not sufficient for gene expression analysis. Despite submitting their manuscript to the journal of agronomy, the authors failed to include any data on physiological, biomass, yield, or other agronomic traits. That is really disappointing. Therefore, the inclusion of several agronomic and biochemical traits by the authors significantly strengthens the manuscript. I don’t have any interest in rejecting the manuscript. I advised authors to try to include some agronomic data and clarify my doubts mentioned below.

Response: Thank you very much for your thoughtful review of our manuscript and your valuable suggestions for improvement. We truly appreciate your insights. The primary objective of our study was to conduct a gene expression analysis in leaf tissue, as we believe this focus is significant for understanding the regulatory mechanisms involved in wheat stress responses. While we fully acknowledge the importance of physiological and agronomic trait analyses, we chose to concentrate on gene expression because previous studies, such as Zhang et al. (2022), have extensively addressed these traits in similar contexts. Our aim was to provide new insights into the functioning of TaGLKs under stress conditions through a focused bioinformatics and gene expression approach. We are grateful for your suggestion to include physiological and agronomic data. Although this study emphasizes gene expression, we are considering how to incorporate such analyses in future research to enhance our findings further. We believe that our approach offers significant contributions to the field and aligns well with the objectives of the Journal of Agronomy. In response to your kind advice and detailed suggestions, we have revised the manuscript accordingly. We hope these revisions meet both your expectations and the journal's standards.

Reference:

Zhang G et al. Molecular and phenotypic characterization of Chinese wheat (Triticum aestivum) cultivars for resistance to Fusarium head blight. Plant Breeding, 2022, https://doi.org/10.1111/pbr.13055.

 

 

 

1. The authors should change the tile because they did not characterize the genes. They simply analysed gene expression analysis.

Response: Thank you for your time to review our manuscript. We have revised the title to reflect the focus of our study on gene expression analysis according to your kind suggestion: “Identification and Expression Analysis of Wheat Golden2-like (TaGLK) Gene in Response to Biotic and Abiotic Stress”.

2) In the abstract, authors should include the original results instead of bioinformatics analysis results.

Response: Thank you for your insightful comment. We have revised the abstract according to your kind advice.

3. Is GLK Genes or TF? Kindly clarify and use the same pattern throughout the manuscript.

Response: Thank you for your comment. GLK is a transcription factor, we have revised the manuscript to clarify and ensured consistent terminology throughout.

4. At position 60, which disease resistance genes does GLK TF induce?

Response: Thank you for reviewing our manuscript. The disease resistance genes induced by GLK TFs are the Cucumber mosaic virus resistance genes in Arabidopsis. We have revised the manuscript to clarify it.

5. On page 77, the authors noted that C-Repeat Binding Factor (CBF) transcription factors stimulate GLK genes. However, in L. No. 60 and other sections of the manuscript, the authors mention that GLK transcription factors induce disease resistance genes. I'm really confused. The authors should first clarify whether GLK is a gene or a transcription factor.

Response: Thank you very much for your time to review our manuscript, we have revised the manuscript to reflect that GLK is a transcription factor.

6. Indeed, researchers have implicated several other candidate genes in enhancing biotic and abiotic stress tolerance in wheat, in addition to GLK genes/TF. Therefore, the authors should elucidate what distinguishes GLK genes from other genes. Why is it necessary to focus on GLK genes? It's crucial to emphasize this in the introduction section, as it elevates your study above existing reports.

Response: Thank you for your insightful comment. We have revised the introduction to clearly articulate what distinguishes GLK genes from other candidate TFs implicated in stress tolerance in wheat. We have emphasized their unique regulatory roles and potential contributions to enhancing plant resilience. This focus on GLK genes highlights the significance of our study in advancing understanding of stress responses in wheat beyond existing reports: “Previous studies have reported that TFs such as WRKY, NAC, MYB, and GRF are associated with various processes, including seed development, leaf senescence, plant proliferation, expansion, and responses to biotic and abiotic stresses [55]. However, the regulatory function of GLKs in plant stress responses remains poorly understood. Although some research has indicated that GLKs play a role in regulating stress-responsive pathways in specific plant species, the precise mechanisms by which they contribute to stress tolerance in wheat are still unclear. This study investigated the regulatory roles of TaGLK TFs under various stresses, including osmotic stress, ABA treatment, and infection by F. graminearum, using bioinformatics, gene expression data, and RT-qPCR analyses to better understand the potential regulatory role of TaGLKs in wheat during abiotic and biotic stress conditions. The findings offer essential insights for further exploration of the roles of TaGLKs and their possible application in the molecular breeding of stress-tolerant wheat cultivars”.

7. Sentences are not necessary for Nos. 96-97, as the authors collected the sequences directly from phytozome databases.

Response: Thank you for your precious time and insightful comment, we have revised the manuscript according to your kind advice.

8. The authors should specify in Section 2.1 which version of the wheat genome they used to mine genes from the phytozome database.

Response: Thank you for your insightful comment. We have revised Section 2.1 to specify the version of the wheat genome “Triticum aestivum cv. Chinese Spring v2.1” used for mining TaGLKs from the Phytozome database.

9. How did the authors provide the gene names for Wheat GLK?

Response: Thank you for your insightful question. We provided the gene names for TaGLKs based on the common wheat abbreviation ("Ta"), followed by "GLK" and their physical position on the chromosomes. We have clarified this at the Section 3.2 in the revised manuscript to ensure transparency in our methodology; “The TaGLKs were renamed based on the common wheat abbreviation ("Ta"), followed by "GLK" and their physical positions (from lowest to highest) on the chromosomes, ranging from 1A to Un (Figure 2)”.

10. Authors used only 40 GLK of rice and 10 GLK of Arabidopsis protein sequences to retrieve GLK genes for wheat. However, both plants do not possess more than 40 GLK proteins. Then how did authors collect the remaining GLK gene sequences for wheat? Authors should clarify and include these details in the materials and methods section.

Response: Thank you very much for your time to review our manuscript and your insightful comments and questions for improvement. We would like to kindly clarify from Section 2.1 that the protein sequences used to search for the TaGLK proteins were from “tobacco (LOC107817975), Arabidopsis (AT2G20570.2 and AT5G44190.1), maize (LOC542493), and rice (LOC4326363)”. The previously reported 46 rice GLKs by [Bhutia et al., 2020] and 10 Arabidopsis GLKs by [Alam et al., 2020] sequences obtained from the Phytozome v13 database were used for phylogenetic analysis of the wheat GLKs.

Reference:

Bhutia, K.L; Nongbri, E.L.; Gympad, E.; Rai, M.; Tyagi, W. In silico characterization, and expression analysis of rice golden 2-like (OsGLK) members in response to low phosphorous. Molecular biology reports, 2020, 47(4), 2529–2549.

Alam, I.; Wu, X.; Yu, Q.; Ge, L. Comprehensive Genomic Analysis of G2-like Transcription Factor Genes and Their Role in Development and Abiotic Stresses in Arabidopsis. Diversity 2022, 14, 228.

1. No. 110: Phytozome provides the chromosome number of any given gene. Why did the authors use the GFF3 file to determine the chromosomal location of the GLK gene? Kindly clarify this. What did you mean by the GFF file in this context?

Response: Thank you for your constructive feedback and for highlighting an important aspect of our methodology. We utilized the GFF3 file primarily because it provides detailed annotations and features for the entire genome, including gene models, functional elements, and their chromosomal locations. While Phytozome does indeed provide chromosome numbers for genes, the GFF3 file offers a comprehensive framework for understanding the relationships and organization of genes within the context of the wheat genome. In this study, we referred to the GFF3 file to ensure we captured all relevant genomic features associated with the TaGLKs, including their precise locations and any additional annotations that could inform our analysis. We have revised the manuscript to provide a clearer explanation of our approach: “Using the wheat genome GFF3 file obtained from the Phytozome v13 database, the distribution of the TaGLKs across the wheat chromosomes was visualized using MapInspect v1.0.”

12. The authors should alter the title of Section 2.6, as those experiments are solely focused on bioinformatics.

Response: Thank you for your insightful comment, we have revised the title of the section 2.6 according to your kind suggestion.

13. In Section 2.6, how does PEG600 differ from drought? The study also explored the impact of PEG600-induced drought stress. What is the selected stress basis for this experiment?

Response: Thank you for your thoughtful review of our manuscript and for allowing us the opportunity to clarify this important concept. Regarding the abiotic stress treatments, we obtained gene expression data from the WheatOmics 1.0 database. Our study includes distinct drought and heat treatments, as well as combined drought and heat stress in 2 different wheat cultivars, which are separate from the PEG6000 treatment in another wheat cultivar (Figure 6B). We chose this approach to comprehensively investigate how TaGLKs respond to both drought (PEG6000-induced) and heat conditions, as well as to PEG6000 treatment only in different wheat cultivars. This distinction is important for understanding the regulatory mechanisms of TaGLKs in response to varying stress environments in wheat.

14) NO. 165l; Jingshuang 16 cultivar resistance What abitoic tress? drought or cold or heat?

Response: Thank you for your insightful comments regarding the characterization of the cultivar. While the cultivar is known for its moderate susceptibility to powdery mildew and stripe rust, we acknowledge that some studies have reported its resilience to certain abiotic stresses such as drought, high temperature, and salt. However, to maintain clarity and accuracy in our method, we have revised the manuscript to focus solely on the biotic stresses (moderately susceptible to powdery mildew and stripe rust) to ensure that our explanations are firmly grounded in the established understanding of the cultivar performance.

15. The phylogenetic analysis revealed a close relationship between the majority of TaGLK proteins. How did the authors claim his? So, authors should reconstruct the phylogentic tree whose plant contains around 125 GLK proteins.

Response: Thank you for reviewing our manuscript and suggestions for improvement. Regarding the phylogenetic tree, 125 TaGLKs, 46 OsGLKs, and 10 AtGLKs were used. Based on the conserved domain analysis using the NCBI-CDD database, the results revealed that TaGLK members in each of the 6 subfamilies of the phylogenetic tree contain the Myb-like DNA binding domain and the SANT domain (Supplementary figure S1). We have revised the discussion section to provide a clear explanation for this statement: “Phylogenetic analysis indicated that all six subfamilies of the TaGLK contain members that possess the conserved Myb-like DNA binding domain and the SANT domain. This suggests that these TaGLK subgroups may share similar functional roles in DNA binding and the regulation of gene expression”.

16. Figure 4 displays only 99 GLK. Where are the results for the remaining 26 proteins?

Response: Thank you for your insightful comments. With regards to figure 4, all the 125 TaGLKs were including in the figure but not orderly arranged from TaGLK1 through TaGLK125. We have revised figure 4 to clearly arrange them from TaGLK1 through TaGLK125 to enhance clarity and prevent any potential ambiguity.

17. Which are the foundational genes chosen for the gene expression analysis experiment?

Response: Thank you very much for your time to review or manuscript. 8 TaGLKs were chosen for RT-qPCR gene expression based on the expression levels of the TaGLKs from the WheatOmics 1.0 database in Section 2.6: “Based on the expression profiles, 8 expressed TaGLKs (TaGLK4, TaGLK28, TaGLK32, TaGLK36, TaGLK55, TaGLK59, TaGLK98, and TaGLK117) in biotic and abiotic stress treatments were chosen for RT-qPCR gene expression study under abiotic and biotic stress treatments”.

18. The authors should include the temperature and product size of the primers used for gene expression analysis.

Response: Thank you for your insightful suggestion, we have revised the supplementary file 1 to include the primers temperature and product size based on your kind advice.

19. Authors should include the position or location of each gene in the supplemental file, which will help identify whether authors found different genes or the same locus. This should be more important.

Response: Thank you once again for your time to review our manuscript. We have revised the supplementary file 3 to include the chromosome number and gene positions according on your kind suggestion.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Xiang and colleagues provides an in-depth bioinformatic analysis of the Wheat Golden2-like gene (TaGLK) family, elucidating its functional characteristics in response to biotic and abiotic stress conditions. The conditions analyzed include osmotic stress, abscisic acid treatment, and infection by Fusarium graminearum. Using computational analysis of available datasets, the authors identified approximately 125 TaGLK genes that exhibit differential regulation in response to these environmental cues. Furthermore, they analyzed various characteristics of these genes, such as amino acid length, protein stability, and subcellular localization. The study revealed that these genes are distributed across different chromosomes, and a conserved motif analysis identified the presence of at least 20 motifs within the gene family. Functional annotations, including gene ontology (GO) and molecular function analysis, highlighted the involvement of TaGLKs in processes such as development, stress tolerance, and metabolism. Lastly, the authors validated their findings by experimentally examining the expression patterns of select TaGLK genes under biotic and abiotic stress conditions, uncovering significant differences in expression. Overall, the study provides valuable insights into the TaGLK gene family and their potential roles in wheat development and stress tolerance.

 

Overall, this manuscript presents a well-executed bioinformatic investigation of the TaGLK gene family, complemented by experimental validation. The study is logically structured, supported by clear figures, and well-written. Below, I provide a few minor comments and suggestions for improvement. By addressing these minor issues, the manuscript will further improve its clarity and scientific rigor.

 

1.        The authors examined the expression patterns of eight TaGLK genes under biotic and abiotic stress conditions. However, the rationale for selecting these specific genes is not clearly explained. Providing criteria for their selection would enhance the clarity of this section.

2.        The methodology section mentions that the data represents relative expression. However, the manuscript does not specify the reference gene used for normalization. Including this information is essential for reproducibility.

3.        It is recommended to annotate bar graphs with “ns” (non-significant) where applicable, to clarify statistical comparisons.

4.        In the infection experiment figure, the X-axis is labeled as “(d),” whereas the text refers to “(h).” This inconsistency should be corrected to avoid confusion.

5.        In the first half of the manuscript, the authors repeatedly state that TaGLKs are involved in “stress responses” (e.g., lines 232, 299, 312). However, at this stage of the manuscript, no experimental evidence supports this claim. The conclusions appear to be based on analyses of amino acid length, chromosome rearrangement, and other structural features, which are not direct indicators of stress response function. This discrepancy should be addressed to ensure the claims are substantiated by the data presented.

6.        Only 6 out of 80 annotated TaGLK genes are linked to water deprivation (line 348). Yet, the authors claim that stress response is a major function of TaGLKs (line 352). This conclusion seems to overlook the diverse roles of TaGLKs, including seed development (involving 18 genes). The authors should rephrase their conclusions to reflect the broader functional spectrum of TaGLKs.

 

 

Author Response

This manuscript by Xiang and colleagues provides an in-depth bioinformatic analysis of the Wheat Golden2-like gene (TaGLK) family, elucidating its functional characteristics in response to biotic and abiotic stress conditions. The conditions analyzed include osmotic stress, abscisic acid treatment, and infection by Fusarium graminearum. Using computational analysis of available datasets, the authors identified approximately 125 TaGLK genes that exhibit differential regulation in response to these environmental cues. Furthermore, they analyzed various characteristics of these genes, such as amino acid length, protein stability, and subcellular localization. The study revealed that these genes are distributed across different chromosomes, and a conserved motif analysis identified the presence of at least 20 motifs within the gene family. Functional annotations, including gene ontology (GO) and molecular function analysis, highlighted the involvement of TaGLKs in processes such as development, stress tolerance, and metabolism. Lastly, the authors validated their findings by experimentally examining the expression patterns of select TaGLK genes under biotic and abiotic stress conditions, uncovering significant differences in expression. Overall, the study provides valuable insights into the TaGLK gene family and their potential roles in wheat development and stress tolerance.

 

Overall, this manuscript presents a well-executed bioinformatic investigation of the TaGLK gene family, complemented by experimental validation. The study is logically structured, supported by clear figures, and well-written. Below, I provide a few minor comments and suggestions for improvement. By addressing these minor issues, the manuscript will further improve its clarity and scientific rigor.

Response: Thank you very much for your precious time to review our manuscript and your kind comments for improvement. We have revised the manuscript according to your kind advice and detailed suggestions. We hope that these revisions will satisfy both your expectations and the journal's requirements.

 

1. The authors examined the expression patterns of eight TaGLK genes under biotic and abiotic stress conditions. However, the rationale for selecting these specific genes is not clearly explained. Providing criteria for their selection would enhance the clarity of this section.

Response: Thank you very much for reviewing our manuscript and suggestions. The selection of the 8 TaGLKs for RT-qPCR analysis were based on the genes that were highly expressed in biotic, abiotic stress treatments compared to the control. We have revised the manuscript to include the criteria according to your kind suggestion.

 

2. The methodology section mentions that the data represents relative expression. However, the manuscript does not specify the reference gene used for normalization. Including this information is essential for reproducibility.

Response: Thank you for your insightful comment. The reference gene used for the RT-qPCR normalization is the ADP-ribosylation factor Ta2291. We have revised the manuscript to include the reference gene according to your suggestion: Gene-specific primers of the TaGLKs used for the RT-qPCR analysis (Supplementary file 1) were designed using the Primer3Plus software and the ADP-ribosylation factor Ta2291 was used as internal reference gene.

 

3. It is recommended to annotate bar graphs with “ns” (non-significant) where applicable, to clarify statistical comparisons.

Response: Thank you for your time to review our manuscript. We have revised the manuscript and the figures according to your kind advice.

 

4. In the infection experiment figure, the X-axis is labeled as “(d),” whereas the text refers to “(h).” This inconsistency should be corrected to avoid confusion.

Response: Thank you for your insightful comment on the figure 9. We have revised the figure and change the Time (d) to Day (d) to accurately describe the treatment period based on your kind advice.

 

5. In the first half of the manuscript, the authors repeatedly state that TaGLKs are involved in “stress responses” (e.g., lines 232, 299, 312). However, at this stage of the manuscript, no experimental evidence supports this claim. The conclusions appear to be based on analyses of amino acid length, chromosome rearrangement, and other structural features, which are not direct indicators of stress response function. This discrepancy should be addressed to ensure the claims are substantiated by the data presented.

Response: Thank you for your review and insightful suggestions. We have revised the manuscript to clearly explain the results according to your kind suggestion to avoid any ambiguity in the results.

 

6. Only 6 out of 80 annotated TaGLK genes are linked to water deprivation (line 348). Yet, the authors claim that stress response is a major function of TaGLKs (line 352). This conclusion seems to overlook the diverse roles of TaGLKs, including seed development (involving 18 genes). The authors should rephrase their conclusions to reflect the broader functional spectrum of TaGLKs.

Response: Thank you once again for your insightful feedback. We have revised the manuscript to include the diverse functions of the TaGLKs according to your advice: “These findings indicate that TaGLKs play significant roles in wheat development processes, particularly through their involvement in various biological functions, including photosynthesis, transpiration, and regulation of seed development under stress conditions”.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors addressed all the comments properly and improved the content of the manuscript according to my suggestion.