Transcriptome Differential Expression Regulation Analysis of the Narrow-Leaf Mutant of Sorghum Bicolor

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper offers a clear analysis of 1,520 genes that are expressed differently and linked to the narrow-leaf trait, giving important information about how genes are regulated in Sorghum bicolor. The techniques are robust, and the results sound. In addition, the research addresses a critical aspect of plant morphology that can directly impact crop yield, making it relevant to agronomic practices and potential breeding programs.

I have some suggestions for improvement: The introduction is very limited in scope. It would be helpful to include a more detailed look at past research on narrow-leaf mutants in other crops such as rice and maize, which would give a better understanding of why the findings are important. Integrate a more thorough examination of the existing literature on narrow-leaf mutants and their physiological implications to contextualize the findings better.

Also in the discussion, the authors make broad assertions about the implications for crop improvement without adequately discussing potential limitations or challenges in applying these findings in practice.

Figure 11 is presented without statistics to check the significance of the observed differences. Please include a student's t-test or similar.

Clarify the methodology. Some methods lack detailed descriptions. Ensure that all experimental procedures are described in sufficient detail to allow for replication, including growth conditions, statistical analyses, and methodology for DEG identification.

Finally, the paper uses different formats for abbreviations (e.g., DEGs, WT). Please use consistent formatting throughout the paper to improve clarity.        

Author Response

Comments 1：The introduction is very limited in scope. It would be helpful to include a more detailed look at past research on narrow-leaf mutants in other crops such as rice and maize, which would give a better understanding of why the findings are important. Integrate a more thorough examination of the existing literature on narrow-leaf mutants and their physiological implications to contextualize the findings better.

Response1: Thank you for your feedback. We have added relevant content to the introduction, including additional discussions on existing plant hormone research to better align with the context. The revised sections are from line 55- 90.

Comments 2：Also in the discussion, the authors make broad assertions about the implications for crop improvement without adequately discussing potential limitations or challenges in applying these findings in practice.

Response2: Thank you for your valuable input. We have added relevant content to the Discussion section. The revised portion is from lines 386- 392.

Comments 3：Figure 11 is presented without statistics to check the significance of the observed differences. Please include a student's t-test or similar.

Response3: Thank you for your correct and valuable suggestions. We have added the corresponding content in the relevant sections as required. The revised part is from lines 383- 384.

Comments 4：Clarify the methodology. Some methods lack detailed descriptions. Ensure that all experimental procedures are described in sufficient detail to allow for replication, including growth conditions, statistical analyses, and methodology for DEG identification.

Response4: Thank you for your suggestions. We have added corresponding content to the Materials and Methods section.  The revised part is from lines 121- 123.Every piece of your advice has improved our manuscript.

Comments 5：Finally, the paper uses different formats for abbreviations (e.g., DEGs, WT). Please use consistent formatting throughout the paper to improve clarity.

Response5: Thank you for your suggestions. We have rechecked the abbreviation formats throughout the manuscript and corrected some errors. Your dedication is greatly appreciated, and your feedback has been extremely helpful for improving our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Regarding the introduction to the manuscript, the authors describe in detail the role of leaf morphology in rice and maize in providing photosynthetic assimilants to plants, but the topic of the manuscript is completely different: “ Transcriptome differential expression regulation analysis of 2 the narrow-leaf mutant of Sorghum bicolor”.

Unfortunately, these issues, as well as hormone synthesis and hormonal regulation of plants, were left out of the authors' consideration in the introduction.

Regarding the section of research results presented in Supplementary Figure 4, they are not available to the reader of the journal, they cannot be recognized, read and analyzed to determine the authors' contribution to the formation of this figure.

I recommend that the authors revise this figure, possibly by dividing it into several figures, so that the information presented in the figure would be accessible to the reader of the journal.

 

Next, let's look at how to enter the title of a paper in the References list:

Ma, H.; Bell, K.N.; Loker, R.N. Qpcr and Qrt-pcr analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies. Mol. Ther. Methods Clin. Dev. 2021, 20, 152–168,

Firstly, the title does not correspond to the original article, which in my opinion is unacceptable and needs to be corrected, and secondly, this is a review of clinical trials of gene therapy IN HUMANS, and there is no mention of plants at all... Obviously, the authors have not read this review, or the link was accidentally included in the list of references. In any case, it should be removed and the relevant experimental article performed on plants should be cited.

 

Regarding the references to the article at number 46, line 289, I cannot consider it successful, there are a number of more recent articles and I suggest that the authors of the manuscript replace this reference.

            I would like to draw the attention of the authors to the fact that their reference to the article at number 48 does not correspond to the content of the reference, .this article is about Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice, and I did not find information about the biosynthesis of plant hormones, as the authors write, lines 309-310.

 

The authors' attention should be drawn to the fact that glycosylation converts free, active forms of cytokine bases into an inactive form, and does not affect the process of cis-zeatin synthesis at all, as the authors write: “Sobic.010G238400 and Sobic.006G174300 are involved in encoding cis-zeatin O-beta-D-glucosyltransferase, and all three genes are involved in the synthesis pathway of the important transferase cis-zeatin O-beta-D-glucosyltransferase and UDP (uridine diphosphate). The expression of these genes was downregulated in nal6 leaves, and thus they may have a direct effect on the synthesis of cis-zeatin”, lines 314-318.

 

I cannot help but draw the authors' attention to the fact that their references are not correct:

The authors found that the DEGs were mainly concentrated in two pathways, phytohormone signaling and phenylpropane biosynthesis, and exogenous spraying of gibberellin partially restored the growth of the short-stalked, narrow-leaved mutant at [54,55], lines 348-350 are not appropriate and would contradict their statement about the role of gibberellins. For example, article number 54 states:

“The role of cis-zeatin-type cytokinins in plant growth regulation and mediating responses to environmental interactions”, lines 563-565.

As for the article at number 55, it is not accessible to a number of modern search databases that I have used, I recommend that the authors replace this article with one that is accessible to modern search systems.

 

An annoying inaccuracy in the reference to the authors' statements: “The results showed that DEGs were mainly enriched in pathways such as plant hormone biosynthesis, signal transduction, and cell wall biosynthesis, in which more than 100 DEGs were related to the synthesis and signaling of  phytohormones, such as IAA, GA, ABA, ETH, and BR”, at n: 56,57. The first article deals only with the role of cytokines, which the authors do not cite in their citation, lines 353-356.

56.Terceros, G.C.; Resentini, F.; Cucinotta, M.; Manrique, S.; Colombo, L.; Mendes, M.A. The importance of cytokinins during reproductive development in arabidopsis and beyond. Int. J. Mol. Sci. 2020, 21, 8161, doi:10.3390/ijms21218161., lines 569- 570

 

The other article deals only with the exogenous effect of auxin on sorghum mesocotyl elongation.:

57.Liu C, Yao Z, Jiang B, Yu W, Wang Y, Dong W, Li Y, Shi X, Liu C, Zhou Y. Effects of exogenous auxin on mesocotyl elongation of sorghum. Plants (Basel). 2023, 12, 944. doi: 10.3390/plants12040944, lines 571-572.

 

In my opinion, the authors should carefully correct all inaccuracies

Comments on the Quality of English Language

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

Author Response

Comments 1: Regarding the introduction to the manuscript, the authors describe in detail the role of leaf morphology in rice and maize in providing photosynthetic assimilants to plants, but the topic of the manuscript is completely different: “ Transcriptome differential expression regulation analysis of 2 the narrow-leaf mutant of Sorghum bicolor”.

Unfortunately, these issues, as well as hormone synthesis and hormonal regulation of plants, were left out of the authors' consideration in the introduction.

Response1: Thank you for you piont out.In the introduction section, we aim to highlight the necessity of our research by describing the significance of leaf morphology in sorghum breeding. However, studies on sorghum leaf morphology are extremely limited. Therefore, we intend to emphasize the research significance by referencing studies on leaf morphology in other gramineous crops.

Additionally, your suggestions regarding plant hormone synthesis and signal transduction have been invaluable for revising our manuscript. As a result, we have incorporated this content into the introduction.

The revised sections are from line 55 - 90.

Comments 2: Regarding the section of research results presented in Supplementary Figure 4, they are not available to the reader of the journal, they cannot be recognized, read and analyzed to determine the authors' contribution to the formation of this figure.

I recommend that the authors revise this figure, possibly by dividing it into several figures, so that the information presented in the figure would be accessible to the reader of the journal.

Response2: Thank you for pointing out the issue, which has been extremely helpful for improving the figures! In response to your suggestions, we have revised Supplementary Figure 4. Specifically, we removed pathways irrelevant to the manuscript and added heatmaps of corresponding DEGs  within the pathways. This adjustment aims to help readers more intuitively and clearly identify the DEGs showing changes.

Comments 3: Next, let's look at how to enter the title of a paper in the References list:

Ma, H.; Bell, K.N.; Loker, R.N. Qpcr and Qrt-pcr analysis: Regulatory points to consider when conducting biodistribution and vector shedding studies. Mol. Ther. Methods Clin. Dev. 2021, 20, 152–168,

Firstly, the title does not correspond to the original article, which in my opinion is unacceptable and needs to be corrected, and secondly, this is a review of clinical trials of gene therapy IN HUMANS, and there is no mention of plants at all... Obviously, the authors have not read this review, or the link was accidentally included in the list of references. In any case, it should be removed and the relevant experimental article performed on plants should be cited.

Response3: We sincerely apologize for the oversight. Thank you for your meticulous review and valuable feedback. We have updated the references as follows:

48. Spartz AK, Lee SH, Wenger JP, Gonzalez N, Itoh H, Inzé D, Peer WA, Murphy AS, Overvoorde PJ, Gray WM. The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion.Plant J. 2012 Jun;70(6):978-90. doi: 10.1111/j.1365-313X.2012.04946.x.

The revised sections are from line 630 - 632.

Comments 4: Regarding the references to the article at number 46, line 289, I cannot consider it successful, there are a number of more recent articles and I suggest that the authors of the manuscript replace this reference.

Response4: Thank you for your suggestions regarding the literature citation section. We have revised the references as follows:

49. Gajdosová S, Spíchal L, Kamínek M, Hoyerová K, Novák O, Dobrev PI, Galuszka P, Klíma P, Gaudinová A, Zizková E, Hanus J, Dancák M, Trávnícek B, Pesek B, Krupicka M, Vanková R, Strnad M, Motyka V. Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J Exp Bot. 2011May;62(8):2827-40. doi: 10.1093/jxb/erq457.

The revised sections are from line 636 - 639.

Comments 5: I would like to draw the attention of the authors to the fact that their reference to the article at number 48 does not correspond to the content of the reference, .this article is about Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice, and I did not find information about the biosynthesis of plant hormones, as the authors write, lines 309-310.

Response5: Thank you for you piont out. We have updated the references as follows:

51  Thilakarathne AS, Liu F, Zou Z. Plant Signaling Hormones and Transcription Factors: Key Regulators of Plant Responses to Growth, Development, and Stress. Plants (Basel). 2025 Mar 31;14(7):1070. doi: 10.3390/plants14071070.

The revised sections are from line 645 - 647.

Comments 6: The authors' attention should be drawn to the fact that glycosylation converts free, active forms of cytokine bases into an inactive form, and does not affect the process of cis-zeatin synthesis at all, as the authors write: “Sobic.010G238400 and Sobic.006G174300 are involved in encoding cis-zeatin O-beta-D-glucosyltransferase, and all three genes are involved in the synthesis pathway of the important transferase cis-zeatin O-beta-D-glucosyltransferase and UDP (uridine diphosphate). The expression of these genes was downregulated in nal6 leaves, and thus they may have a direct effect on the synthesis of cis-zeatin”, lines 314-318.

Response6: We have carefully analyzed your suggestions and fully agree with your perspective. In response, we have revised the corresponding sections and updated all related parts of the manuscript. Thank you for your valuable input, which has been instrumental in improving our work.

The revised sections are from line 357 - 368.

Comments 7: I cannot help but draw the authors' attention to the fact that their references are not correct:

The authors found that the DEGs were mainly concentrated in two pathways, phytohormone signaling and phenylpropane biosynthesis, and exogenous spraying of gibberellin partially restored the growth of the short-stalked, narrow-leaved mutant at [54,55], lines 348-350 are not appropriate and would contradict their statement about the role of gibberellins. For example, article number 54 states:

“The role of cis-zeatin-type cytokinins in plant growth regulation and mediating responses to environmental interactions”, lines 563-565.

As for the article at number 55, it is not accessible to a number of modern search databases that I have used, I recommend that the authors replace this article with one that is accessible to modern search systems.

Respons7: Thank you for your help, which is of great importance. The 54th reference does not match the relevant content, so we have removed it to ensure the standardization of literature citation.

The 55th reference is from a Chinese journal and can be retrieved on CNKI (China National Knowledge Infrastructure). I have attached the article as proof of the authenticity of our citation.

Once again, we sincerely appreciate your assistance!

Comments 8: An annoying inaccuracy in the reference to the authors' statements: “The results showed that DEGs were mainly enriched in pathways such as plant hormone biosynthesis, signal transduction, and cell wall biosynthesis, in which more than 100 DEGs were related to the synthesis and signaling of  phytohormones, such as IAA, GA, ABA, ETH, and BR”, at n: 56,57. The first article deals only with the role of cytokines, which the authors do not cite in their citation, lines 353-356..

56.Terceros, G.C.; Resentini, F.; Cucinotta, M.; Manrique, S.; Colombo, L.; Mendes, M.A. The importance of cytokinins during reproductive development in arabidopsis and beyond. Int. J. Mol. Sci. 2020, 21, 8161, doi:10.3390/ijms21218161., lines 569- 570

The other article deals only with the exogenous effect of auxin on sorghum mesocotyl elongation.:

57.Liu C, Yao Z, Jiang B, Yu W, Wang Y, Dong W, Li Y, Shi X, Liu C, Zhou Y. Effects of exogenous auxin on mesocotyl elongation of sorghum. Plants (Basel). 2023, 12, 944. doi: 10.3390/plants12040944, lines 571-572.

Respons8: Your suggestions are very correct, and we appreciate your input. We have revised the two references as follows:

58. Han, L.; Jiang, C.; Zhang, W.; Wang, H.; Li, K.; Liu, X.; Liu, Z.; Wu, Y.; Huang, C.; Hu, X. Morphological Characterization and Transcriptome Analysis of New Dwarf and Narrow-Leaf (dnl2) Mutant in Maize.Int. J. Mol. Sci. 2022, 23, 795. https://doi.org/10.3390/ijms23020795
59. Wang, B.; Smith, S.M.; Li, J. Genetic regulation of shoot architecture. Annu. Rev. Plant Biol.2018, 69, 437–468.

The revised sections are from line 666 - 673.

Author Response File: Author Response.pdf