BnaMYB73, a Brassica napus L. R2R3-MYB Transcription Factor, Enhances Plant Salt and Osmotic Stress Tolerance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

I evaluated the paper under reference plants-4121226.

The paper presents a contribution to understanding the role of BnaMYB73 in osmotic stress tolerance in colza.  The authors have adopted a rigorous methodology combining transcriptional, physiological and molecular analyses. The paper is well-structured, and the results are generally relevant and of good quality.

However, it requires minor revisions before its publication.

• The abstract should be revised to emphasize the most significant findings. The authors should indicate the p-values for the reported findings.
• Mannitol is used to simulate osmotic stress. However, at high concentration, it should be noted that it can also act as an osmoprotectant, helping to stabilize cellular proteins and membranes and scavenging reactive oxygen species. So, PEG is generally more relevant than mannitol (just for your consideration in future work).
• During germination, seedlings do not yet accumulate new biomass through photosynthesis; therefore, this parameter has limited significance at this stage. However, it may be retained as a complementary indicator alongside other germination-related parameters.
• Given the large volume of data, it would be helpful to provide more detailed and precise figure titles to enhance clarity and make the results easier to follow.
• Regarding the units of SOD and POD, if possible, I would prefer referring the enzyme activity to mg of proteins.
• In your case, do you consider proline as a tolerance marker or as a stress indicator?
• Regarding Actin genes used as the reference genes. Did you validate that these genes are stable under your stress conditions? Actin is commonly used, but under salt/osmotic stress its expression can sometimes vary.

Conclusion: The results provide new insights into the role of BnaMYB73 in enhancing traits associated with osmotic stress tolerance. The findings support the initial hypothesis and highlight BnaMYB73 as a promising candidate for developing stress-tolerant rapeseed varieties.

Recommendation: Accept with minor revisions.

Comments for author File: Comments.pdf

Author Response

Point by point response to Reviewer #1

 

General Comments

The paper presents a contribution to understanding the role of BnaMYB73 in osmotic stress tolerance in colza. The authors have adopted a rigorous methodology combining transcriptional, physiological and molecular analyses. The paper is well-structured, and the results are generally relevant and of good quality. However, it requires minor revisions before its publication.

Response: Thank you for your comments.

 

Main comments:

1. The abstract should be revised to emphasize the most significant findings.

Response: Thank you. We have revised the abstract to emphasize the most significant findings, as suggested.

 

2. The authors should indicate the p-values for the reported findings.

Response: Thank you. We have indicated the p-values for the reported findings in Fig. 2C of the revised manuscript.

 

3. Mannitol is used to simulate osmotic stress. However, at high concentration, it should be noted that it can also act as an osmoprotectant, helping to stabilize cellular proteins and membranes and scavenging reactive oxygen species. So, PEG is generally more relevant than mannitol (just for your consideration in future work)

Response: Thank you. We will use PEG to simulate osmotic stress in future work.

 

4. During germination, seedlings do not yet accumulate new biomass through photosynthesis; therefore, this parameter has limited significance at this stage. However, it may be retained as a complementary indicator alongside other germination-related parameters

Response: Thank you for your comment. During germination, seedlings do not accumulate new biomass through photosynthesis, so the cotyledon greening rate only primarily reflects plant’s sensitivity to abiotic stress at this stage. Therefore, we have retained it as a complementary indicator alongside other germination-related parameters.

 

5. Given the large volume of data, it would be helpful to provide more detailed and precise figure titles to enhance clarity and make the results easier to follow.

Response: Thank you for your suggestion. We have updated the figure titles in Figure 3- 6 to be more detailed and precise, thereby enhancing clarity and making the results easier to follow in the revised manuscript.

 

6. Regarding the units of SOD and POD, if possible, I would prefer referring the enzyme activity to mg of proteins.

Response: Thank you. The units of SOD and POD activities were kept consistent with the measurement method used. As the activities were calculated based on the fresh weight of the samples rather than protein concentration, we retained the current units to ensure the interpretability and comparability of the data.

 

7. In your case, do you consider proline as a tolerance marker or as a stress indicator?

Response: Thank you. In our case, we consider proline both as a tolerance marker and as a stress indicator. As a stress indicator, proline accumulation is a well-known response to various stresses, including salt, osmotic, and drought stress, reflecting stress severity and cellular metabolic changes. In our study, BnaMYB73 upregulation under salt and osmotic stress conditions was associated with increased proline levels, which highlights its role as an indicator of stress intensity (Fig. 2D, E; Fig. 6D). On the other hand, proline also serves as a marker of stress tolerance. As shown in seed germination and seedling growth assays (Fig. 3, Fig. 4), transgenic plants expressing BnaMYB73 displayed enhanced salt and osmotic stress tolerance, which correlated with increased proline accumulation. Proline protects cells by stabilizing proteins, membranes, and reducing oxidative damage (Fig. 6), thereby improving the plant’s ability to withstand stress. Thus, proline plays a dual role in our study, both reflecting stress response and facilitating stress tolerance.

 

8. Regarding Actin genes used as the reference genes. Did you validate that these genes are stable under your stress conditions? Actin is commonly used, but under salt/osmotic stress its expression can sometimes vary.

Response: Thank you for your comment. We have validated that BnaActin expression remains generally stable under salt and osmotic stress conditions, supporting its use as a reference gene. As shown in Fig. 2D and 2E, we performed a semi-quantitative assay for the BnaActin7 gene using all cDNA templates treated with 150 mM NaCl and 20% PEG6000. The figure below showed that BnaActin7 expression was generally stable across all samples.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "BnaMYB73, a rapeseed R2R3-MYB transcription factor, enhances plant salt and osmotic stress tolerance" reported the research of identifying and characterizing a member of the R2R3-MYB subfamily, BnaMYB73 in a rapeseed variety.  Function analysis of BnaMYB73 were conducted, which proved BnaMYB73 functions as a stress-responsive transcription factor that displayed significantly enhanced tolerance to salt and osmotic stress. This research is well designed, and the results strongly support the conclusions. Manuscript is very well organized; pictures and tables are very clear.  This is a high quality manuscript and I recommend accepting it.

Author Response

Point by point response to Reviewer #2

General Comments

The manuscript entitled "BnaMYB73, a rapeseed R2R3-MYB transcription factor, enhances plant salt and osmotic stress tolerance" reported the research of identifying and characterizing a member of the R2R3-MYB subfamily, BnaMYB73 in a rapeseed variety. Function analysis of BnaMYB73 were conducted, which proved BnaMYB73 functions as a stress-responsive transcription factor that displayed significantly enhanced tolerance to salt and osmotic stress. This research is well designed, and the results strongly support the conclusions. Manuscript is very well organized; pictures and tables are very clear. This is a high quality manuscript and I recommend accepting it.

Response: Thank you for your positive comments and encouragement.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript explores the function of BnaMYB73, an R2R3-MYB transcription factor from Brassica napus, and shows that its overexpression can improve salt and osmotic stress tolerance when introduced into Arabidopsis thaliana. The work is well organized and brings together bioinformatic analysis, molecular assays, physiological measurements, and whole plant phenotyping to build a consistent story around the role of BnaMYB73 as a positive regulator of abiotic stress responses. The authors provide convincing evidence that BnaMYB73 localizes to the nucleus, acts as a transcriptional activator, is strongly induced by salt and PEG treatments in rapeseed, and that its heterologous expression enhances germination, seedling growth, adult plant performance, antioxidant capacity, proline accumulation, and the expression of well-known stress-responsive genes such as RD29B, DREB2A, RAB18, P5CS1, SOS1, and CAT1. The addition of yeast assays, physiological analyses, and evaluation across multiple developmental stages adds depth to the dataset.

However, the study is largely descriptive and relies almost entirely on overexpression in Arabidopsis. There is a lack of functional validation in rapeseed itself, and also no loss-of-function approach to complement the overexpression data. In addition, the manuscript does not provide mechanistic evidence to show whether BnaMYB73 directly regulates the downstream stress-responsive genes whose expression is reported. Some methodological details and statistical descriptions are not sufficiently clear.  Several figures would benefit from improved presentation, and the English language needs careful polishing for clarity and precision. Overall, the results are promising and relevant, but the manuscript would benefit from clearer methodological reporting, a more critical discussion, and language refinement before it can be considered ready for publication.

 

Author Response

Point by point response to Reviewer #3

General Comments

This manuscript explores the function of BnaMYB73, an R2R3-MYB transcription factor from Brassica napus, and shows that its overexpression can improve salt and osmotic stress tolerance when introduced into Arabidopsis thaliana. The work is well organized and brings together bioinformatic analysis, molecular assays, physiological measurements, and whole plant phenotyping to build a consistent story around the role of BnaMYB73 as a positive regulator of abiotic stress responses. The authors provide convincing evidence that BnaMYB73 localizes to the nucleus, acts as a transcriptional activator, is strongly induced by salt and PEG treatments in rapeseed, and that its heterologous expression enhances germination, seedling growth, adult plant performance, antioxidant capacity, proline accumulation, and the expression of well-known stress-responsive genes such as RD29B, DREB2A, RAB18, P5CS1, SOS1, and CAT1. The addition of yeast assays, physiological analyses, and evaluation across multiple developmental stages adds depth to the dataset.

Response: Thank you for your comments.

 

Main comments:

1. However, the study is largely descriptive and relies almost entirely on overexpression in Arabidopsis. There is a lack of functional validation in rapeseed itself, and also no loss-of-function approach to complement the overexpression data. In addition, the manuscript does not provide mechanistic evidence to show whether BnaMYB73 directly regulates the downstream stress-responsive genes whose expression is reported.

Response: Thank you for this insightful comment. All authors agree that it would be valuable to further elucidate the precise role of BnaMYB73 in rapeseed and to obtain mechanistic evidence demonstrating whether BnaMYB73 directly regulates the downstream stress-responsive genes reported. These aspects are part of our ongoing and future research. We have already generated transgenic Arabidopsis lines and are currently developing transgenic rapeseed plants constitutively expressing BnaMYB73 for further functional validation and mechanistic evidence.

In the present work, we mainly focused on evaluating whether heterologous expression of BnaMYB73 in Arabidopsis could enhance salt and osmotic stress tolerance. Since both Arabidopsis and rapeseed belong to the Brassicaceae family, the well-established Arabidopsis system is widely used for preliminary functional characterization of genes from rapeseed. Similar experimental designs, published in the journal Plants, have also been employed in many studies, as shown below.

Niu, J., Bai, J., Sun, S., Fan, Y., Li, J., Cao, Y., Li, L., Jin, H., Zhang, L., Meng, F., & Luo, Q., 2026. Overexpression of AmCML24 improves abiotic stress tolerance and upregulates stress-responsive genes in Arabidopsis. Plants, 15(3), 420.

Su, X., Wei, J., Cui, J., Sun, X., Ma, J., Bi, Z., Liu, Y., Liu, Z., Zhao, Y., Li, Y., Zhao, F., Bai, J., Yao, P., & Sun, C., 2026. Heterologous expression of the StCML50 gene enhances drought tolerance in Transgenic Arabidopsis. Plants, 15(3), 417.

Min, T., Zuo, Y., Manda, T., Li, Y., Lu, Y., Xu, H., Chen, J., & Yang, L., 2026. LhSBP1 gene of liriodendron hybrid enhances the cold resistance of plants by regulating ROS metabolism. Plants, 15(2), 196.

Xiao, X., Zhou, H., Yu, P., Zheng, W., Han, D., Yang, L., Jiang, Z., Cheng, Y., Li, Y., Huang, T., Xiong, W., Huang, X., Chen, M., Liu, X., Zhang, M., Huang, Y., & Zhou, Q., 2026. Overexpression of BnaMATE43b improves resistance to aluminum toxicity and identification of its upstream transcription factors in Rapeseed (Brassica napus L.). Plants, 15(2), 338.

Yu, W., Kong, G., Ya, H., & Zhang, H., 2025. Overexpression of hevea brasiliensis HbCDS2 gene enhances cold tolerance in Transgenic Arabidopsis. Plants, 14(23), 3591.

Han, G., Ren, K., He, R., Mo, R., Zeng, J., & Sui, M., 2025. Ectopic over-expression of BjuAGL9-2 promotes flowering and pale-yellow phenotype in Arabidopsis. Plants, 14(22), 3502.

Guo, M., Li, S., Tian, J., Li, M., Zhu, X., Guo, C., & Shu, Y., 2025. TrWRKY41: A WRKY transcription factor from white clover improves cold tolerance in Transgenic Arabidopsis. Plants, 14(22), 3493.

Sun, G., He, S., Dong, J., He, T., Zhu, X., Wang, K., Zhang, Z., Liu, C., Hong, L., & Xing, J., 2025. PoMPK3, an MAPK gene from Purslane (Portulaca oleracea), conferred salt tolerance in Transgenic Arabidopsis thaliana. Plants, 14(22), 3478.

Yang, X., Wang, K., Guan, F., Shi, B., Xie, Y., Du, C., Tang, T., Yang, Z., Ma, S., & Wan, X., 2025. The Bitter Gourd Transcription Factor McNAC087 Confers Cold Resistance in Transgenic Arabidopsis. Plants, 14(22), 3440.

Zhuang, W., Sun, L., An, J., Zhu, J., Yan, T., Wang, T., Shu, X., & Wang, Z., 2025. Ectopic expression of a poplar gene PtrMYB119 confers enhanced tolerance to drought stress in Transgenic Nicotiana tabacum. Plants, 14(21), 3251.

Wang, J., Niu, J., Hu, M., Chen, M., Li, X., Song, Z., Yin, S., Zhu, F., Jiao, J., Tang, R., Wang, F., Li, R., & Li, H., 2025. Overexpression of the lipid transfer protein gene SpLTP1 from Desert Pioneer Plant Stipagrostis pennata Enhances the Drought Tolerance in Arabidopsis. Plants, 14(20), 3198.

 

2. Some methodological details and statistical descriptions are not sufficiently clear.

Response: Thank you. We have carefully revised the Materials and Methods and statistical analysis sections to provide clearer and more detailed descriptions. The corresponding changes have been incorporated into the revised manuscript.

 

3. Several figures would benefit from improved presentation, and the English language needs careful polishing for clarity and precision.

Response: Thank you for your suggestion. Several figures, including Fig. 2, Fig.5, Fig. 6 and Fig. 7, have been improved for better presentation. In addition, the manuscript has undergone language editing to improve clarity, precision, and academic rigor.

 

4. Overall, the results are promising and relevant, but the manuscript would benefit from clearer methodological reporting, a more critical discussion, and language refinement before it can be considered ready for publication.

Response: Thank you for your constructive comments. We have thoroughly revised the manuscript to address your concerns. Specifically, the Materials and Methods section has been carefully revised to provide clearer and more detailed descriptions. The Discussion has been expanded to include a more critical interpretation of our findings. In addition, the entire manuscript has been carefully edited to improve the clarity and precision of the language.