Uncovering Hierarchical Regulation among MYB-bHLH-WD40 Proteins and Manipulating Anthocyanin Pigmentation in Rice

Round 1

Reviewer 1 Report

Sun et al. report the hierarchical regulation of bHLH, MYB, and WD40 involved in the anthocyanin regulation in rice. Several mutant and overexpression lines were analyzed to construct a model of the regulatory system. Overall, the study is of interest to the community. Please see my specific comments below.

1) The title needs modification: The anthocyanin pigmentation is not modified through a discovery. Someone would need to act based on the discovered insights.

2) Anthocyanin biosynthesis genes should be the same in all organs. Listing various plant parts in the abstract suggest that there would be differences. The only difference might be their regulation.

3) Transport of the anthocyanins should not be neglected (see https://doi.org/10.3390/plants11070963). This is often the crucial factor when engineering anthocyanin pimentation. Vacuolar pH changes could be an additional factor (see https://doi.org/10.3389/fpls.2020.580085).

4) Anthocyanins require the addition of sugar moieties to anthocyanidins. Some additional details about the decoration/modification should be included (e.g. Toghe et al., 2005).

5) Transcriptional regulation of anthocyanin biosynthesis is not so conserved between monocots and dicots. For example, have a look at the banana MYBs: there are two clades which might be involved in the process (https://doi.org/10.1371/journal.pone.0239275, Fig.2). In contrast, dicots like Arabidopsis have only one (MYB75/MYB90/MYB113/MYB114).

6) Were PC NIL and PH NIL not characterized in the previous study?

7) Is the MBW complex required for anthocyanin biosynthesis activation in all organs of rice? If I remember correctly, some monocots can activate the anthocyanin biosynthesis just based on MYB and bHLH.

8) The WA1 selection should be based on the phylogenetic tree and not on a BLAST hit. Also, it is important to include additional sequences. An outgroup like Marchantia or Amborella is missing.

9) Fig2B seems to have redundant information: identify and divergence do not have to been shown. One of them should be enough.

10) Fig.2D: I would suggest to use the same colors for orthologs across all species. This seems not to be the case for the B3 DNA binding protein.

11) WA1 overexpression leads to lower anthocyanin content in the hulls. The authors discuss this as a potential repression, but I could also imagine an activation of competing pathways. Is it possible that the proanthocyanidin (PA) content is increased? The activation of PA biosynthesis was not investigated in this study, but could contribute to the coloration.

12) Overexpression/KO of WA1 is not proof that this gene activates the anthocyanin biosynthesis genes directly. It is also possible that it just activates activators of the anthocyanin biosynthesis. The authors might want to phrase this more carefully.

13) The subcellular localization is not surprising. I would suggest to move this into the supplements.

14) WD40 proteins were previously considered as scaffolding proteins that connect bHLH and MYB. Please include these references in the discussion.

15) Please include sequences of all plasmids in the supplements.

16) The method section would generally benefit from additional details.

17) All datasets must be included in the supplements.

18) The language quality is unacceptable. This needs to be improved systematically.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Authors submitted a manuscript entitled “Manipulating anthocyanin pigmentation by uncovering hierarchical regulation among MYB-bHLH-WD40 proteins in rice” to the International Journal of Molecular Sciences. In this work, they identified the culm-specific pigmentation gene S1 through map-based cloning. They mentioned that its alleles are also known for hull/pericarp pigmentation. They found that one WD40 protein-encoding gene- WA1 is essential for anthocyanin biosynthesis in rice. Further, they investigated that, the cascading regulation between MBW members, S1 (bHLH) acts as the master gene by activating the expression of C1 (MYB), and then C1 activates the promoter of WA1 (WD40). They concluded that MBW members coordinate in a common way to efficiently regulate anthocyanin biosynthesis genes. The findings presented in this paper will help rice breeders to design new rice varieties with anthocyanin accumulation in specific organs. This paper falls under the aims and scopes of this journal and can be considered after some revisions.

Whether the author found genes which showed functional redundancy with target genes?

Fig 7 B, C; The information about callus induction is missing. Please provide the methodology for callus induction. How can you say that callus showed anthocyanin production? Have you done any analysis regarding this or only saying based on appearance?

The discussion section is week. Please speculate the results and discuss them in relevance to previous literature and based on your findings. Moreover, conclude and provide me sentences on research gaps and possible future outlines. Discussion is not acceptable. It needs substantial improvements.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript provides new data on the culm-specific pigmentation gene S1 and clarified that one WD40 protein encoding gene WA1 is essential for anthocyanin biosynthesis in rice. Therefore, based on the obtained data, the authors consider what could be the essential genes for anthocyanin biosynthesis in rice organs, with the idea of building the knowledge and the foundations to produce a rice that accumulate anthocyanin in specific organs by transferring endogenous genes. The topic is certainly very important and current also considering the ever-growing demand for health products and the enormous consumption of rice in the world.

As a general observation, citation and reference seem congruent with the topic and with journal format.

Introduction

Is well written and offers enough background for readers. Nevertheless, it seems to be quite long especially in comparison to the discussion. I recommend reviewing it and considering whether some parts could be brought into the discussion.

Also, some minor comments:

As you will see in the note in the manuscript, I suggest introducing some bibliographical references in the first part.

Results

In general, the results as well as the entire manuscript are well written, the reading is very clear and fluid.

The greatest difficulty I encountered while reading, especially this part, was the use of acronyms without specifying them in full. Probably for those who work with this species they are clear, but for those who, like me, work with other species it is useful to specify better. I have inserted some notes in the text where I think it is useful to specify. The same is true for Figure S3.

Discussion

Is presented very well and the only thing, that I already mentioned, it could be considered to pass some parts of the introduction here.

Materials and Methods

Molecular procedures, laboratory handling and statistical approach are very detailed, and readers can trace them easily. I compliment the authors on how they dealt with such laborious and detailed procedures, and I have no suggestions to improving it.

Finally, I have only one observation to make, some parts of this manuscript are very similar to those of the paper "Sun, X .; Zhang, Z .; Chen, C .; Wu, W .; Ren, N .; Jiang, C .; Yu, J .; Zhao, Y .; Zheng, X .; Yang, Q. et al. The C-S-A gene system regulates hull pigmentation and reveals evolution of anthocyanin biosynthesis pathway in rice. J. Exp. Bot. 2018, 69 (7): 1485-1498 ", which partly includes the same authors of this manuscript, and which is cited several times by the authors.

I congratulate the authors for their work and my recommendation is to accept the manuscript for publication after including these minor suggestions.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors have addressed the comments and revised the manuscript accurately. I think it is suitable for publication. Moreover, I request authors to do some experiments on callus anthocyanin accumulation. Your current results based on callus phenotype are not satisfactory.