From Structure to Function of Promoters and 5′UTRs in Maize

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review summarizes the structural organization of promoters and 5' untranslated regions (5'UTRs) in maize, along with their general roles in gene expression. However, it primarily focuses on describing these structural features, with limited discussion on their specific functions. To enhance its impact, the manuscript could be improved in the following ways:

1. Expand the functional analysis: The summary should integrate discussions on how these regulatory elements control gene expression with specific mechanisms affecting maize traits, such as yield and stress resistance.
2. Incorporate some summary tables: some tables should be added to systematically present the structures of key promoters and 5'UTRs, along with their experimentally verified functions. 

Author Response

Response to Reviewer 1 comments

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

This review summarizes the structural organization of promoters and 5' untranslated regions (5'UTRs) in maize, along with their general roles in gene expression. However, it primarily focuses on describing these structural features, with limited discussion on their specific functions. To enhance its impact, the manuscript could be improved in the following ways:

1. Expand the functional analysis: The summary should integrate discussions on how these regulatory elements control gene expression with specific mechanisms affecting maize traits, such as yield and stress resistance.

We thank the reviewer for the suggestion. Where possible, we added practical data regarding functional impact. However, in many cases, direct links between mechanisms and function remain speculative. The chosen review topic aims to present current understanding of basic principles, while also acknowledging the lack of strong, systematic functional validation. One of the goals of this review is to highlight potential theoretical and experimental opportunities, even in one of the most extensively studied monocot model plants.

2. Incorporate some summary tables: some tables should be added to systematically present the structures of key promoters and 5'UTRs, along with their experimentally verified functions.

We thank the reviewer for the suggestion. We added two tables: one summarizing promoters and the other summarizing 5′ UTR elements. In the promoter table, we present a list of experimentally validated promoters in maize, classified according to their key functional types. In the 5′ UTR table, we systematically summarize the key elements described to date, highlight maize-specific features, and include the applications of each element to emphasize not only their function but also their practical relevance.

Response to comments on the quality of English language

We have revised the grammar and attached the updated version of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General assessment

The paper examines the significant issue of how promoters and 5′UTRs collaboratively influence gene expression in maize and how these components can be manipulated for agricultural enhancement. The manuscript is informative and well-referenced; however, the text is occasionally dense and might benefit from enhanced clarity through improved language, smoother transitions, and the inclusion of integrative components such as figures or tables, along with a better "take-home" message in important sections.

Major comments

1. Clarify scope and novelty in the Introduction

In the last paragraph of the introduction, make it clear what is different from earlier promoter/UTR reviews. For example, you could say that you are focusing on maize as a monocot model, combining chromatin-/enhancer-level data with translational control, and highlighting machine-learning-guided design.

You might want to add one or two words explaining why maize is a great system for testing promoter–5′UTR combinations, such as its strong transformation pipelines and large genomic resources.

2. Balance and tighten the Promoters vs 5′UTR sections
   • The promoter section, which includes subsections 2.1 and 2.2, is very long and very detailed. The 5′UTR part, on the other hand, is well written but does not feel quite as connected to the engineering point of view.
   • To make the paper easier to read, you might want to cut down on the parts that explain things in great detail (like long lists of motif classes and TF families) and replace them with a summary table that says things like "Common cis-elements in maize promoters, position, and example functions."
   • Including a short section or paragraph that clearly compares the relative roles of promoter engineering and 5′UTR engineering in transgene expression tunability in maize, giving specific examples of how the 5′UTR changed output greatly even though the promoter stayed the same.
3. Strengthen the “Applications for genetic engineering” narrative
   • Section 2.2 lists a lot of examples of synthetic, constitutive, tissue-specific, and inducible promoters, but it sounds more like a catalog than anything else.
   • Ideas for making things better: In the last paragraph of section 2.2, add a short "guidelines" paragraph that lists some practical rules for picking promoters in maize, such as when to use ZmUbi1 vs. OsAct1 vs. virus-derived promoters, when tissue-specific or inducible promoters are better to avoid pleiotropy, and common mistakes like silencing of repeated CaMV 35S sequences. Draw more attention to how the choice of promoter affects construct design (for example, gene stacking, bidirectional promoters, multiplex traits) and present regulatory issues (for example, viral promoters and public perception, though this needs to be phrased carefully).
4. Clarify mechanistic distinctions (IRES vs CITE; insulators; non-canonical caps)
   • The parts about IRES, CITEs, and non-canonical caps are helpful, but they might be hard for people who are not experts to understand.
   • Please summarize in one to two lines what has been shown to be true in maize vs. what has been extrapolated from Arabidopsis or other systems, focusing on IRES, CITE, and NAD+ caps.
   • In the insulator/silencer discussion, it should be made clearer that there is still disagreement about what plant insulators do and that new work on small DNA elements suggests behavior that depends on the situation rather than canonical insulators in the sense of metazoans.
5. Add at least one summary/informational figure or table for 5′UTRs
   • Figure 2 is useful but still quite schematic.
   • A small table with typical features of maize 5′UTRs (average length, enrichment of uORFs in certain functional classes, presence/absence of Cap1/Cap2, typical intron positions, etc.) and their qualitative impact on translation would help readers apply this information in construct design.

Author Response

Response to Reviewer 2 comments

Thank you very much for such a deep and thoughtful review. It was very helpful and we hope we were able to use it well to improve our manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted files.

General assessment

The paper examines the significant issue of how promoters and 5′UTRs collaboratively influence gene expression in maize and how these components can be manipulated for agricultural enhancement. The manuscript is informative and well-referenced; however, the text is occasionally dense and might benefit from enhanced clarity through improved language, smoother transitions, and the inclusion of integrative components such as figures or tables, along with a better "take-home" message in important sections.

Major comments

1. Clarify scope and novelty in the Introduction

In the last paragraph of the introduction, make it clear what is different from earlier promoter/UTR reviews. For example, you could say that you are focusing on maize as a monocot model, combining chromatin-/enhancer-level data with translational control, and highlighting machine-learning-guided design.

You might want to add one or two words explaining why maize is a great system for testing promoter–5′UTR combinations, such as its strong transformation pipelines and large genomic resources.

We thank the reviewer for the suggestion. We’ve added this clarification to highlight the differences and the value of the review for potential readers.

2. Balance and tighten the Promoters vs 5′UTR sections

The promoter section, which includes subsections 2.1 and 2.2, is very long and very detailed. The 5′UTR part, on the other hand, is well written but does not feel quite as connected to the engineering point of view.

To make the paper easier to read, you might want to cut down on the parts that explain things in great detail (like long lists of motif classes and TF families) and replace them with a summary table that says things like "Common cis-elements in maize promoters, position, and example functions."

Including a short section or paragraph that clearly compares the relative roles of promoter engineering and 5′UTR engineering in transgene expression tunability in maize, giving specific examples of how the 5′UTR changed output greatly even though the promoter stayed the same.

We thank the reviewer for the suggestion and want to clarify the goals that we aimed for in this section. We included a detailed characterization of sequence-level elements because, in our view, the vast majority of existing review articles lack a thorough analysis of recent insights, particularly in monocots and maize. In many reviews, key cis-regulatory elements, their functions, and their genomic locations are presented primarily in tables, which can lead to a loss of biological context due to oversimplification. We acknowledge that this section may appear slightly outside the main scope; however, we believe that it represents an important layer of regulatory control. Moreover, this huge section can serve as an accessible entry point to the topic and as a foundation for generating new ideas for bioinformatics and machine-learning specialists.

We fully agree with your comment regarding the need to compare the relative roles of promoter engineering and 5′UTRs. We addressed this point in the Conclusions section, as we consider it the appropriate place to synthesize the separately discussed aspects of promoters and 5′UTRs. We also revised the text to clarify and specify the reported ~10-fold effect of different 5′UTRs observed in maize experiments.

3. Strengthen the “Applications for genetic engineering” narrative

Section 2.2 lists a lot of examples of synthetic, constitutive, tissue-specific, and inducible promoters, but it sounds more like a catalog than anything else.

Ideas for making things better: In the last paragraph of section 2.2, add a short "guidelines" paragraph that lists some practical rules for picking promoters in maize, such as when to use ZmUbi1 vs. OsAct1 vs. virus-derived promoters, when tissue-specific or inducible promoters are better to avoid pleiotropy, and common mistakes like silencing of repeated CaMV 35S sequences. Draw more attention to how the choice of promoter affects construct design (for example, gene stacking, bidirectional promoters, multiplex traits) and present regulatory issues (for example, viral promoters and public perception, though this needs to be phrased carefully).

We thank the reviewer for the suggestion. To provide a concise overview of experimentally validated promoters in maize, we added a table classifying these promoters according to their key functional types. In addition, we have updated the Conclusions section to include guidance, as suggested by the reviewer.

4. Clarify mechanistic distinctions (IRES vs CITE; insulators; non-canonical caps)

The parts about IRES, CITEs, and non-canonical caps are helpful, but they might be hard for people who are not experts to understand.

Please summarize in one to two lines what has been shown to be true in maize vs. what has been extrapolated from Arabidopsis or other systems, focusing on IRES, CITE, and NAD+ caps.

In the insulator/silencer discussion, it should be made clearer that there is still disagreement about what plant insulators do and that new work on small DNA elements suggests behavior that depends on the situation rather than canonical insulators in the sense of metazoans.

We thank the reviewer for highlighting this. We have revised the text to better reflect the uncertainty and ongoing debate surrounding the insulator/silencer discussion. 

Because IRES, CITE, and NAD⁺ represent functionally distinct mechanisms, we chose to emphasize maize-specific insights within each corresponding topic. We hope that this approach addresses your suggestion and improves the clarity of the text for the reader.

5. Add at least one summary/informational figure or table for 5′UTRs

Figure 2 is useful but still quite schematic.

A small table with typical features of maize 5′UTRs (average length, enrichment of uORFs in certain functional classes, presence/absence of Cap1/Cap2, typical intron positions, etc.) and their qualitative impact on translation would help readers apply this information in construct design.

We thank the reviewer for the suggestion. We extended the text by adding a 5′UTR table that systematically summarizes the key elements described in the review, highlights maize-specific features, and includes applications of each element to emphasize not only their function but also their practical relevance. We believe that this table will help readers better systematize the detailed information presented in the review.

Response to Comments on the Quality of English Language

We have revised the grammar and attached the updated version of the manuscript.

Author Response File: Author Response.pdf