22KD Zein Content Coordinates Transcriptional Activity during Starch Synthesis in Maize Endosperm

Round 1

Reviewer 1 Report

This revised manuscript was well modified according to the reviewer's comments.

However, the title is too long. I think it needs to simplify by combining your results.

Author Response

We would like to express our appreciation for the time spent on our manuscripts and for your suggestion. We made some changes in the title to better reflect on the outcome of our researches and hope that it will grab reader’s attention.

Regards.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors substantially improved the manuscript.

Some minor changes should be made prior to publication:

Line 4: I believe that there must be some editing error in the title as "of during" does not make any sense. It seems that one word or the other should be deleted. Please check it.

Author Response

We would like to express our appreciation for the time spent on our manuscripts and for your suggestion. We made some changes in the title to better reflect on the outcome of our researches and hope that it will grab reader’s attention.

Regards.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript reports the results and analysis of a transcript profiling experiment comparing 15 DAP endosperms of sh2 and Sh2 in a presumed (see comment 2 below) corn hybrid. Although the use of a hybrid genetic background renders the results less interesting than if more nearly isogenic backgrounds were used as experimental starting material, general trends should still emerge from the transcriptomics.

Overall the research appears well conceived, executed and presented. I did note a few components that should be addressed.

1. The overall design of the experiment is not made clear in the methods or elsewhere in the paper. It should be required that the paper clarifies: (a) what generation was actually sampled for RNA extraction? Was it F2 kernels segregating on the ears of F1 plants, or F3 kernels on the ears of (presumably homozygous) F2 plants?

The term "F3" appears nowhere in the current manuscript so presumably the authors sampled F2 kernels on F1 ears. However the methods section currently states "F2 ears" (which harbor F3 kernels) but elsewhere the term F2 appears more generally.

If the actual tissue was F2 kernels (i.e. F2 endosperms) as best suggested by the current text, it would be imperative to explain how the sh and Sh segregants were identified when the authors collected 15 DAP tissue for RNA extraction. If the actual tissue was F3 kernels, please clarify how the F2 plants were genotyped etc.

2. What is the genetic relationship of HZ508 to B73? Most sweet corn lines are widely divergent from B73. If the two lines are widely divergent, a few points should be addressed:

How did the authors account for differences in read-mapping of B73 vs HZ508 alleles back to the B73 genome? What proportion of HZ508 alleles may not have had transcripts read-mapped under the selected parameters? I suppose there are a number of ways to address the latter question, for example the authors could examine a random collection of polymorphic genes in the low and high expression class, and see if representation is equal for HZ508 and B73 alleles. The same could be done for DEGs.

Although F2 segregants from a wide cross will show enormous genetic variation, the PCA analysis does suggest that 10 endosperms was enough individuals to capture most of the variation.

The authors may also wish to note that since sweet corn is typically produced as an inbred, the key functional networks may differ in that genotype from what is reported here.

Author Response

Dear Reviewer：

Thank you for your comments concerning our manuscript entitled “Starch accumulation during endosperm development in maize (Zea mays L.) depends on the activity of transcription factors” (ID: agronomy-729772). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We used the “Track changes” function in Microsoft word and the blue color to highlight all the modifications in the resubmitted paper. Our responses to specific comments are detailed in the word file joint to this correspondence.

Regards

Author Response File: Author Response.docx

Reviewer 2 Report

This study used RNA-seq and some analysis approach, showed a network and supported theoretical foundation for starch development, but there are some modifications:

1. Page 3, Line 128-130, if only according to GO analysis results, I think this viewpoint inappropriate, please add more evidence.
2. Figure 2d, please check the Sign position.
3. In Discussion, please check the reference format.

Author Response

Dear Reviewer：

Thank you for your comments concerning our manuscript entitled “Starch accumulation during endosperm development in maize (Zea mays L.) depends on the activity of transcription factors” (ID: agronomy-729772). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We used the “Track changes” function in Microsoft word and the blue color to highlight all the modifications in the resubmitted paper. Our responses to specific comments are available in the word file joint to this correspondence.

Regards.

Author Response File: Author Response.docx

Reviewer 3 Report

OVERALL COMMENTS

With this work, the authors intend to identify, through a transcriptomic coupled to a network analysis approach, key regulators involved in starch biosynthesis in maize kernel. This topic is very important given the importance of starch as the main component of the maize kernel. Moreover, its accumulation is also correlated with the accumulation of other important components such as protein.

Nevertheless, I would like to address some issues that I detected or gave rise to some questions.

TITLE

The title at its present form does not bring anything new to the scientific community. It is already known that transcription factors play a role in the regulation of starch accumulation (e.g. Zhang, Z., Dong, J., Ji, C., Wu, Y., & Messing, J. (2019). NAC-type transcription factors regulate accumulation of starch and protein in maize seeds. Proceedings of the National Academy of Sciences, 116(23), 11223-11228.)

Perhaps try to be more specific: Which transcription factors? Any particular family of TFs that weren't previously involved?

The current title does not reflects both the results and the discussion presented below.

ABSTRACT

Except for some minor issues that I have with the last two sentences (see below), the abstract seems fine.  

INTRODUCTION

Although an overview of the shrunken phenotype is given in the Introduction, I believe that an entire paragraph talking on the molecular regulation of sh2 is missing. 

OBJECTIVES

The results presented only partially reflect the objectives proposed by the authors ("we aimed to identify the key regulators of maize kernel development and starch biosynthesis."). I would suggest expanding your objectives to present your results more in accordance with what was initially proposed.   

M&M

Except for some questions I have on some methodologies (see below), I think that overall the M&M are fine.

RESULTS

See comment made in the OBJECTIVES sub-section.

DISCUSSION

I think that the discussion needs some polish. Given the extensive number of results presented before, the length of the DISCUSSION section seems unbalanced.

CONCLUSIONS

Overall, the conclusion lack references as one cannot distinguish between the authors' own work from previous works.

As it stands now, the final conclusions presented in the paper seem to be a little far-fetched since the results are only based on transcriptomic data.

REFERENCES

Different reference formatting styles can be found throughout the manuscript. Please standardize the formatting style used to present the bibliography.

SPECIFIC COMMENTS

ABSTRACT

Line 23 - sh2 mutant lines? (instead of sh2)

Line 26 - "Many of these DEGs": how many? Perhaps give at least a percentage.

Line 29 - "previous work on rice and wheat": although some comparisons are made in relation to rice I could not find in the discussion data that support the similarities between the authors' own results and what is already known in wheat.

Lines 41-42 - Sh2 and Bt2, among other genes (see https://www.maizegdb.org/data_center/reference?id=22566)
Loci (Locus)
bt1 brittle endosperm1
sh1 shrunken1
sh2 shrunken2
sh4 shrunken4

Moreover, I suggest that the authors add information on the type of inheritance of these genes or at least of the shrunken2 (complete dominance?).

Line 50 - "needs" instead of "is"

M&M

Line 62 - Why use line B73?

Line 63 - Why 15 DAP?

Line 63 - the 10 kernels were homozygous? heterozygous? It does not matter?

Line 64 - What is the sample in this context? an ear?

Line 67 - What was pooled? the biological replicates? Please clarify.

Line 82 - How many DEGs were confirmed by qRT-PCR?  

Line 84 - Why use just one reference gene. As far as I know, at least two should be used (Clinical Chemistry, Volume 55, Issue 4, 1 April 2009, Pages 611-622, https://doi.org/10.1373/clinchem.2008.112797)

Line 86 - Why put the information about the software package in the sub-heading?

Line 89 - What is SRA? Reference?

Line 89 - What is "row"? Perhaps the authors mean raw?

Line 93 - WGCNA, what does this mean? Reference?

Line 94 - In which assumptions is this network constructed. Please provide some more details.

RESULTS

Line 100 - Which 3 libraries? This is not described in the M&M section. Please add it.

Line 111 - "padj" what does this mean?

Line 114 & Line 120 - sh2 mutant lines instead of "sh2"

Line 132 - Figure 1 need some editing, In its current form it is difficult to read, especially the (a) panel.

Line 153 - "some metabolites": which metabolites? Please clarify. I thought that the authors were talking only about zeins and starch...

Line 168 - Why surprisingly? I would expect that starch as one key component of carbon partitioning would influence many of the carbon metabolism pathways. (https://doi.org/10.1093/jxb/erx291)

Line 168 - "Many DEGs": how many? Try not to be so relative in your statements. Quantify the changes (e.g. give a percentage)

Line 172 - "and" instead of "And"

Lines 185-186 - Did the authors measure the osmotic pressure? If not a reference is missing.

Lines 254-255 - The role of TFs in kernel development and starch biosynthesis is already known. Instead of "suggest" perhaps "supports"; and given that references should be added to this sentence this type of interpretation should be in the Discussion section instead of the Results section.

Lines 291-292 - This sentence is repeated in lines 254-255.

Line 299 - The correlations coefficients and p-values are not visible.

Line 299 - No genes name are shown in the rows.

Line 301 - The network connections are not visible. We cannot read the names of the genes.

DISCUSSION

Line 303 - The authors should contextualize their results within the developmental stage studied (15 DAP). (e.g. see https://doi.org/10.1016/j.molp.2017.01.008)

Lines 312-313 - "seed increases without increasing or decreasing the percentage of starch" does not always occur. It is only specific to certain mutations: if the authors want to discuss this fact they should present evidence that the sh2 mutation used in this work is the same as the mutation referred to in ref 6 in which "The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. "

Line 315 - "similar to what was previously found in" instead of "as revealed in"

Lines 315-317 - Reference is missing.

Lines 318-319 - Reference is missing.

Line 337 - Can you explain how did you get to the conclusion that the "knocking down of Sh2 induces physiological stress"? In sh2 mutant the many the expression of genes involved in stress response is triggered. No stress metabolite was measured to enable the authors to make such a statement.

Lines 338-339 - These conclusions are about which results? Only regarding the results on the stress response genes or to the other up- or down-regulated genes? Please clarify.

Lines 341-342 - Are all the 357 TF DEG key-regulators? Please clarify.

Line 344 - What does the "orange module" stands for in practical terms? What does it represents?

Author Response

Dear Reviewer：

Thank you for your comments concerning our manuscript entitled “Starch accumulation during endosperm development in maize (Zea mays L.) depends on the activity of transcription factors” (ID: agronomy-729772). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We used the “Track changes” function in Microsoft word and the blue color to highlight all the modifications in the resubmitted paper. Our responses to specific comments are available in the word file joint to this correspondence.

Regards.

Author Response File: Author Response.docx