Plant Synthetic Promoters

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors discuss the structure and functions of plant promoters and how the knowledge can be used for synthetic promoter development. Ideas such as using bidirectional promoters for control of multiple genes in transgenics are also discussed.

The article is filled with excellent knowledge and discussion about the plant promoters. The only weak point is the way the article is written, it lacks a flow. Some editing and rewriting to fix the flow would immensely improve the article.

 

Some specific comments:

Line 46: First mention of Figure 1. Figure 1 is referenced at least 8 times before it appears in text. It is usually better to have the figure next to or close to first or second mention. Please check this for other figures as well.

 

Lines 161-168: This paragraph reads like this work was done by the authors instead of describing another paper. The study should be referenced, and text worded properly.

 

Lines 183-186: Is it not the enhancers and other cis-elements that influence the expression pattern and not the core promoter? 

 

Lines 262-269: It is worth noting here that CTCF is unique to bilaterian phyla, and is not found in plants. Since the section is about plant enhancers, it is important to not mix the description with enhancers in general.

 

Figure 2: It is mostly text in large font. It can be made better by reducing text/font size

 

Line 391: what is "they" here?

Lines 458, 466: pathogen?

 

Figure 7 is never referenced in text.

 

Line 679: Deep Learning is first mentioned here, then in line 707 is described as more advanced form of ML, as if describing for the first time.

Line 685: ML models such as

 

Line 704: "Such basic ML approaches" It is unclear which approaches this is referring to, as the lines immediately before are about DL.

 

Line 708: The term Artificial Neural Network (ANN) could be used for describing all types of Neural Networks, but here it is presented as a type of network different from CNN. ANN is an obsolete term, Multi-Layer Perceptron (MLP) is the equivalent type of basic Neural Network. 

 

This whole ML/DL section needs a rewrite.

 

Line 795: What is "OLs" here?

Line 799: FLIP is mentioned once at line 774 in discussion, it is unclear how this line in conclusion is supported.

Author Response

Dear Reviever 1,

Thank you very much for the opportunity to improve the manuscript.

Below are presented detailed responses to all your questions.

Your Sincerely,

Piotr Szymczyk

 

Reviewer 1

Authors discuss the structure and functions of plant promoters and how the knowledge can be used for synthetic promoter development. Ideas such as using bidirectional promoters for control of multiple genes in transgenics are also discussed.

The article is filled with excellent knowledge and discussion about the plant promoters. The only weak point is the way the article is written, it lacks a flow. Some editing and rewriting to fix the flow would immensely improve the article.

 Some specific comments:

1. Line 46: First mention of Figure 1. Figure 1 is referenced at least 8 times before it appears in text. It is usually better to have the figure next to or close to first or second mention. Please check this for other figures as well.

The unnecessary numbers of Figures were removed. Now Figures are close to the localization of the first menton in the text

Fig 1 was removed from line 46.

Figure 1 was transferred to the start of section 2.1, just after the first use in the text.

Fig 2 and 3 were removed from section 3. The bidirectional and orthogonal systems were not precisely characterized here. Fig 2-7 should be transferred to the section 5 .

Changes in Figure numbers: Figures 4 and 5 are now numbered 3 and 4. Figure 3 is now numbered 5.

2. Lines 161-168: This paragraph reads like this work was done by the authors instead of describing another paper. The study should be referenced, and text worded properly.

 The reference [100] was added in two sites in this fragment.

3. Lines 183-186: Is it not the enhancers and other cis-elements that influence the expression pattern and not the core promoter? 

You are generally right. However in the presented fragment, referenced in [103] Authors analysed the expression data in context of core promoter structure. Therefore it was described in this way to be consitent with presented results.

4. Lines 262-269: It is worth noting here that CTCF is unique to bilaterian phyla, and is not found in plants. Since the section is about plant enhancers, it is important to not mix the description with enhancers in general.

The CTCF was removed from the text and Figure 1. The citation [149], concerning CTCF was exchanged to other not related to CTFC and localized a few lines below.

5. Figure 2: It is mostly text in large font. It can be made better by reducing text/font size.

The font size was reduced in Fig 2. The font size in all Figures was the same. I just enlarged the Figure 2 too much. Now  it has the proper, original size and the smaller font size.

6.  Line 391: what is "they" here?

The sentence was corrected to following: As components of synthetic promoters, are typically engaged existing juggled or shuffled oligonucleotide cis-elements.

7. Lines 458, 466: pathogen?

Yes, it should be pathogen not patogen. Corrected in the text.

8. Figure 7 is never referenced in text.

The Figure 7 is referenced in section 5.

9. Line 679: Deep Learning is first mentioned here, then in line 707 is described as more advanced form of ML, as if describing for the first time.

The fragment referring to DL was removed, together with the citation.

Reviewer 2 Report

Comments and Suggestions for Authors

Title

Synthetic plant promoters

Abstract

In the end, a perspective (usefulness) of the review would be missing.

In general, a well-structured, clear summary with the necessary information. It would only be necessary to address the topic, description, types, etc. more. of synthetic promoters.

Keywords

Excess keywords

1. Introduction

Lines 29-32 are 10 quotes to support that idea?

Lines 32-34 Not only through transgenesis is plant resistance to biotic or abiotic factors achieved.

In general, the introduction contains more information on the disadvantages found when using synthetic promoters than the advantages.

2. Vegetable Promoter Sections

Lines 114-125 is the description of the same work, Amack et al. (2023). It would be better to synthesize the information (address more the results than the methods).

This entire section is based on the description of studies related to plant promoters. However, a review that can contribute something different to those already on the network should mention the possible causes of these phenomena based on the available literature (see the example in lines 174-177).

Lines 183-185 it is not appropriate to contrast a previous study with the same study.

Line 214 More significantly?

Lines 245-260 only describe previous studies without trying to give a scientific explanation.

5. Orthogonal expression systems

Line 572-577 cite the developer of the new technique

6. Support for machine learning and deep learning for synthetic promoter preparation

Line 688, so many appointments are necessary

7. Discussion

Lines 725-726 If there are already several studies on promoter synthesis, how is this review different? Just the year of completion? I think this is the opportunity to justify the reason for the revision.

Lines 726-727 For what purpose?

New line 730? In what sense. Could it be with outstanding agronomic characteristics?

In general, a good section addressed what was raised in the review. However, the pre-conclusion is reached that although the topic of the synthesis of synthetic promoters has advanced excessively in recent years, we still do not fully understand the phenomenon. This is indicated at the beginning of the review, therefore the contribution of this review is not seen.

8. Conclusions and future directions

With what purpose?

 

The manuscript addresses a current topic of global interest. However, gathering information from a large number of previous studies does not produce a higher quality review than one that contains few references. The quality of the reviews is based on trying to explain with scientific support the why of the phenomena that are studied? (clearly based on the literature). The manuscript cannot be accepted in its current form, minor corrections must be addressed.

 

Author Response

Dear Reviever 2,

Thank you very much for the opportunity to improve the manuscript.

Below are presented detailed responses to all your questions.

Your Sincerely,

Piotr Szymczyk

 

Reviewer 2

1. Title

Synthetic plant promoters

2. Abstract

In the end, a perspective (usefulness) of the review would be missing.

In general, a well-structured, clear summary with the necessary information. It would only be necessary to address the topic, description, types, etc. more. Of synthetic promoters.

The abstract was modified according to suggestions. Following fragment was added in the final Abstract section:

The broader application of novel, synthetic promoters reduces the frequency of homology-based gene silencing or improves the stability of transgenes.  

Among them are presented orthogonal systems based on transcription activator-like effectors (dTALEs), synthetic dTALE activated promoters (STAPs) and dCas9-dependent artificial trans-factors (ATFs).

Synthetic plant promoters are a valuable tools to provide the precise spatio-temporal regulation and introduction logic gates into complex genetic traits, that is important for basic research studies and their applications in crops plant development. Exactly regulated metabolic routes are less prone to undesirable feed-back regulation and energy waste, improving the efficiency of transgenic crops.

3. Keywords

Excess keywords            

The excess of keywords is removed. Following keywords were left: plant synthetic promoter, orthogonal systems, bidirectional promoters

1. Introduction
2. Lines 29-32 are 10 quotes to support that idea?

The number of citations was reduced to 7.

1. Lines 32-34 Not only through transgenesis is plant resistance to biotic or abiotic factors achieved.

Following sentence was added with proper citations: Plant genetic modifications support other methods increasing plant resistanc to biotic and abotic stresses as crops breeding and priming [24,25].

III. In general, the introduction contains more information on the disadvantages found when using synthetic promoters than the advantages.

Advantages of plant synthetic promoters were provided in the final part of Introduction, following fragment was added:

Despite these limitations, studies on plant synthetic promoters provide valuable information of regulatory mechanism controlling gene expression in complex traits, enabling the regulation of metabolic pathways activity [28,50,78]. Moreover, of particular importance is research on plant bidirectional and orthogonal expression systems, providing solutions to problems associated with coordinated and controlled expression of stacked genes as well as decreasing the exposition to homologous DNA sequences [28,78,79]. Besides the putative practical applications, studies on plant synthetic promoters are often performed in the whole genome scale, supported by advanced machine and deep learning algorithms. These studies supply essential and novel information concerning regulatory functions of promoter segments or cis-active motifs in context of DNA or chromatin structure modifications [35,44,46,50,78].

2. Vegetable Promoter Sections
3. Lines 114-125 is the description of the same work, Amack et al. (2023). It would be better to synthesize the information (address more the results than the methods).

Results are now addressed more than methods. The fragment is corrected to the following one: The importance of the TATA-box sequence for proper gene expression regulation was confirmed by Amack et al. [89]. The authors used PCR and specific primers to introduce single nucleotide substitutions to the TATA-box of the CaMV35S promoter. Two obtained variants, A4T and T5A, showed respectively 1.2 and 1.1-fold higher activity in plant protoplasts as compared to the wild-type TATA-box sequence. Moreover, eight mutants (T1G, A4C, A4G, T5A, A6G, A7C, A7G, and A7T) presented similar or higher activity as the wild-type TATA-box [89].  

1. This entire section is based on the description of studies related to plant promoters. However, a review that can contribute something different to those already on the network should mention the possible the possible causes of these phenomena based on the available literature (see the example in lines 174-177). Several sentences- marked in red were addend to show mechanisms of observed phenomena.

This could be explained by the relative austerity of cis-active motifs, and lower space required to gather them into promoters which reply to limited stimuli.

 Putative mechanism and reasons of presented results were presented. Following fragments were added to section 2.1 to address putative mechanisms of presented results:

The common lack of TATA-box in plant core promoters suggests its putative functional substitution by other core promoter elements as Inr or Y patch, supporting correct DNA-protein interaction during Polymerase II preinitiation complex formation [85,90,96].

The fraction of Y-Patches in plant promoters with distribution peaks around TSS is directionally oriented and methylation sensitive [96]. Moreover, plant promoters with Y-Patch and Inr sequences are also generally stronger than those lacking them, suggesting their putative function in a proper organization of Polymerase II preinitiation complex [85]. Some elements of core promoters observed in humans and D. melanogaster were not found in A. thaliana, such as the Sp1 binding sites or CpG and BRE elements, referring to putative specificity of DNA-protein interactions between plants and animals [96, 98].

The putative functional role of these differences in DNA sequences could be explained by X-ray crystallography studies providing details of DNA-protein interactions in both types of core promoters.

Putatively, the transcription initiation in these promoters is controlled by specific and abundant cis-regulatory elements as well as the open nucleosomal status.

Probably plants GA elements play a role similar to mammalian CpG islands, however GA lements were not methylated [101]. Until now, the significance of presented differences in context of a proper transcription initiation was not studied experimentally.

III. Lines 183-185 it is not appropriate to contrast a previous study with the same study.

It is corrected to not contrast results of the same study. The fragment was corrected as follows:

The association of TATA-box promoters with conditional, regulated gene expression was recently tested [103]. The Coreless promoters indicated a bias towards uniform expression regulation in dicots but not in monocots. A comparison of different plant species found they tended to demonstrate similar expression patterns for a particular promoter. However, the orthologs of the uniformly-expressed genes could be found more easily than those of conditional genes. Moreover, none of the screened core promoter types was consistently associated with changes in gene expression patterns. Therefore, a correlation only occurs between the promoter architecture and expression parameters [103].

1. Line 214 More significantly?

These words were exchanged by the word “Furthermore”. The sentence was corrected as follows: Furthermore, these cis-active motifs are gathered within conserved cis-regulatory modules (CCRM), enabling interactions between trans-factors bound by neighboring, closely-spaced cis-active motifs [67,113,116].

Moreover, following fragment was added to the final part of section 2.2 to explain more mechanistically the functional role of trans-factor complexes organized within proximal promoter:

Transcription factors complexes organized within proximal promoter interacts with Mediator subunits looped towards the RNA Polymerase II preinitiation complex, to affect its activity, represented by gene transcription rate [34]. Among examples of such interactions is MED25 subunit physically associating with the basic helix-loop-helix transcription factor MYC2 in promoter regions of MYC2 target genes to positively affects the MYC2-regulated gene expression. However, the binding of MED25 to the basic Leu zipper transcription factor ABA-INSENSITIVE5 (ABI5) in promoter regions of ABI5 target genes shows a negative effect on ABI5-regulated gene transcription [120]. Moreover, MED18 subunit interacts with SUPPRESSOR OF FRIGIDA4 and ABA INSENSITIVE 4 to regulate flowering time and abscisic acid responses, respectively [121]. 

1. Lines 245-260 only describe previous studies without trying to give a scientific explanation.

Following fragment was added to give scientific explanation:

Therefore, the activation of enhancer and maintenance of its active state is a coordinated, multistep process, precisely regulated through the recruitment of particular proteins, that are sensitive to molecular signals, modifying their performance. Therefore, the activity of Mediator subunits, affecting the functions of the entire enhancer is regulated by crucial plant phytohormones. Amont them are jasmonie acid (JA) regulating MED25, abscisic acid (ABA) controlling the MED18 or general plant defence processes affecting MED21, MED19a and CKD8  activity [34].

5. Orthogonal expression systems

Line 572-577 cite the developer of the new technique

Citation [28] was added .

6. Support for machine learning and deep learning for synthetic promoter preparation

Line 688, so many appointments are necessary

Two references among five were removed.

7. Discussion

Lines 725-726 If there are already several studies on promoter synthesis, how is this review different? Just the year of completion? I think this is the opportunity to justify the reason for the revision.

Following sentence was added to explain putative differences: Therefore, presentation of the novel research results, combined with individual perspective, could add interesting features to presented studies.

 

Lines 726-727 For what purpose?

Following fragment was added to explain better the purposes of synthetic promoter development:

Presented processes increase demand for genetic modification of plant’s complex metabolic routes, which could be supported by synthetic promoters, enabling precise spatio-temporal regulation of multiple transgenes [153-155,178-180].

New line 730? In what sense. Could it be with outstanding agronomic characteristics?

Yes. The sentence was modified as follows: Plant genetic modification by synthetic promoters is pivotal to introducing metabolic switches, and for enabling the development of novel, more efficient, or stress-resistant crop variants of outstanding characteristics and increased biosynthesis rate of valuable metabolites [1,4,5,9,10].

In general, a good section addressed what was raised in the review. However, the pre-conclusion is reached that although the topic of the synthesis of synthetic promoters has advanced excessively in recent years, we still do not fully understand the phenomenon. This is indicated at the beginning of the review, therefore the contribution of this review is not seen.

Discussion was rewritten to show achievements in the field.

8. Conclusions and future directions

With what purpose?

Section was rewritten to show purposes of research.

 The manuscript addresses a current topic of global interest. However, gathering information from a large number of previous studies does not produce a higher quality review than one that contains few references. The quality of the reviews is based on trying to explain with scientific support the why of the phenomena that are studied? (clearly based on the literature). The manuscript cannot be accepted in its current form, minor corrections must be addressed.

 

Author Response File: Author Response.pdf