Enhancing the expression of ARK1 genes in poplar leads to multiple branches and transcriptomic changes

The ARBORKNOX1 (ARK1) gene is an important gene for regulating plant growth and development; however, transcriptomic responses of enhancing expression of ARK1 gene in poplar are poorly investigated. To provide insight into the gene function of the ARK1 gene in poplar, the ARK1 transgenic poplar ‘717' and ‘84 K' lines were obtained, the morphology of transgenic plants was observed, and transcriptome profiles were compared. The results showed that there were multiple branches in ARK1 transgenic seedlings compared with non-transgenic seedlings. The results of transcriptome analysis showed that there were significant differences in transcriptome profiles between the transgenic lines of ‘717' and ‘84 K', and between non-transgenic lines (CK) and transgenic plants. The real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed the expression levels of the genes involved in the pathway of zeatin biosynthesis and brassinosteroid biosynthesis. The increase in expression levels of AHP and CYCD3 was related to multiple branches. Enhancing the expression of the ARK1 gene in poplar seedlings leads to multiple branches and transcriptomic changes.


1.
The material of "717" transgenic line and the transcriptome data of "717" transgenic line are from the published paper "Over-expression of transcription factor ARK1 gene leads to downregulation of lignin synthesis related genes in hybrid poplar '717'" (Ye et al. Scientific Report, 2020), which is from the same lab of this current manuscript. However, in this manuscript, we could not see any reference or acknowledgement. We are not sure how the publication of these two very related manuscripts is coordinated.
Result 3.1: to declare that the transgenic lines have the phenotype resulted from over expressed ARK1 gene, qPCR data of ARK1 is the most reliable method. The authors also need to Please note that resubmitting your manuscript does not guarantee eventual acceptance, and that your resubmission will be subject to peer review before a decision is made.
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Royal Society Open Science openscience@royalsociety.org on behalf of Dr James Locke (Associate Editor) and Steve Brown (Subject Editor) openscience@royalsociety.org Associate Editor Comments to Author (Dr James Locke): Comments to the Author: Although the work is of interest, the two reviewers both point out major problems with the data and its interpretation. Unfortunately this means we must reject the manuscript.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Major concerns: 1. The plasmid map should be provided in the section of M&M. 2. The confirmation of successfully transgenic events is not satisfied. It needs at least additional Southern blotting not just using PCR only. 3. There is a lack of a control plant (non-transgenic) in Figure 1 for comparing. 4. Figure 1 is out of focus, and the resolution of all figures is not satisfying.
Grammar check: Line54: The KNOX gene family encodes homeobox proteins, which plays an important regulatory role -The KNOX gene family encodes homeobox proteins, which play an important regulatory role Line57: By regulating cell differentiation related to organogenesis -By regulating cell differentiation that related to organogenesis Reviewer: 2 Comments to the Author(s) In the manuscript submitted by Lu et al., the authors characterized the phenotypic and transcriptomic changes in the ARK1 overexpression lines compared to wild type Populous lines. There are some important issues that need to be addressed: Outstanding Comment: 1. The material of "717" transgenic line and the transcriptome data of "717" transgenic line are from the published paper "Over-expression of transcription factor ARK1 gene leads to downregulation of lignin synthesis related genes in hybrid poplar '717'" (Ye et al. Scientific Report, 2020), which is from the same lab of this current manuscript. However, in this manuscript, we could not see any reference or acknowledgement. We are not sure how the publication of these two very related manuscripts is coordinated.
Major Comments: 2. Result 3.1: to declare that the transgenic lines have the phenotype resulted from over expressed ARK1 gene, qPCR data of ARK1 is the most reliable method. The authors also need to show the future readers that what is the promotor for enhancing ARK1 gene? How many transgenic lines did you get? How strong the did the ARK1 express in transgenic lines? How many lines did choose to maintain from qPCR data? What are the reference genes? And so on. The authors declared in the manuscript title that overexpressed ARK1 leads to multiple branches in poplar. But the authors did not document branch numbers. There is no evidence to support the statement. Moreover, in Figure 1, there is no wild type control, no scale bar, and the future readers will not understand how old these seedlings are. 3. Result 3.2: This part has transcriptomic analysis from transgenic "717" and transgenic "84K". The data, for instance, DEG numbers between transgenic "717" and WT "717" are different from it in another paper (Ye et al. Scientific Report, 2020) published by the authors´ lab. The authors did not explain the reason. The Figure 2 could be more informative, for instance, describe what genes are up-regulated and what are down-regulated. 4. Result 3.3: There are thousands of DEGs. It is not clear for us why did the authors choose zeatin, brassinosteroids, phenylpropanoid pathways to give a deeper transcriptomic description? Are they relevant to the branching phenotype? If so, the authors should give evidence or some hypothesis that how those genes would affect those pathways and further affect the phenotype. There are some mistakes, for instance, AHP and CYCD3 they are not in biosynthesis pathways, but in signaling pathways. Figure 3: how did the authors get the maps? From KEGG? The authors marked some up-and down-regulated genes on the maps, but there is not transcriptomic data, which could support the Figure 3. The author should specifically show those genes changed expression level from result 3.2 data. 5. Result 3.4: The data in this part is quite controversial. AHP and CYCD3 are up-regulated according to result 3.3. Why they are not up-regulated in result 3.4, according to Figure 4C? And why the zeatin and brassinosteroids profiles changes in Figure 4A? Figure 4 is quite difficult to read. We recommend the authors re-organize the data in the plots, with clear plot legends and figure legends. This also applies to other figures in this manuscript. 6. The manuscript is not well written enough scientifically and grammarly. The authors did not introduce zeatin, brassinosteroids, phenylpropanoid pathways, either branching studies. And in the discussion, some parts are not very relevant to this manuscript. Lacking reference for some statements is also an issue in this manuscript. Figures are not clear to read. We encourage the authors to re-shape the manuscript and analyze the data more thoroughly and carefully.

Author's Response to Decision Letter for (RSOS-200568.R0)
See Appendix A.

Recommendation?
Accept as is

Comments to the Author(s)
The manuscript has been improved accordingly, and I don't have any further questions.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation? Reject
Comments to the Author(s) In the manuscript resubmitted by Lu et al., the authors characterized the phenotypic and transcriptomic changes in the ARK1 overexpression lines compared to wild type Populous lines. The authors improved this manuscript, but there are still some important issues that need to be addressed: Major Comments: 1.
The quality of the current manuscript is still not good enough. Even though, probably the authors tried to make a small story, it is need to be a qualified story. We do not get why the authors decided to study this ARK1 gene. The authors said that "The ARBORKNOX1 (ARK1) gene is an important gene for regulating plant growth and development; however, its gene network regulation is poorly investigated." There are many genes are important for plant growth. Moreover, the authors did not really studied the network regulation. There are genes either upor down-regulated in the transgenic lines from RNA-seq data. However, any (if not, most genes should be the case) gene is over-expressed in plant, would cause transcriptomic changes. The authors did not study the hierarchical relationship between ARK1 and transcription factors, associated proteins, and their target genes. There are transcriptomic changes in the transgenic plants. But, how does ARK1 affect them?

2.
There are thousands of DEGs in the ARK1 overexpressed lines and there are many pathways or hormones affect branching phenotype in nature. It is still not clear for us why did the authors choose zeatin, brassinosteroids, phenylpropanoid pathways to give a deeper transcriptomic description. The authors listed out some pathways relevant to branching morphology, including stigolactone pathway in the Introduction Section. However, the conclusion was "the researches on zeatin and brassinosteroids signaling pathways and phenylpropanoid biosynthesis pathways should be focused on". Why not strigolactone? This is one example that this manuscript is not scientifically and logically sufficient. There are more.

3.
All the figures are not qualified enough for publication. Need to be made carefully and readable.
Minor Comments: Result 3.1: the authors declared that the ARK1 gene is up-regulated in transgenic lines about 3 times compare to control lines and up-regulated lines had branches about 5 times more control lines. However, there is quantification figures or raw data.

2.
It is good the authors provide plasmid map and performed southern blot as anther reviewer suggested. However, these two figures should be in supplementary figures.

Decision letter (RSOS-201201.R0)
Dear Dr Zhang, On behalf of the Editor, I am pleased to inform you that your Manuscript RSOS-201201 entitled "Enhancing the expression of ARK1 genes in poplar leads to multiple branches and transcriptome profile changes" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers, Associate Editor and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Please note carefully the points of the Associate Editor in responding to the comments of Reviewer 2. It will be important to revise the manuscript accordingly as indicated by the Associate Editor. Therefore, I invite you to respond to the comments and revise your manuscript.
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Additionally, the RNAseq data should be made publicly available before publication. There is currently no result after searching PRJNA633952 in NCBI, which is the reference given for the dataset.
Reviewer comments to Author:

Reviewer: 1 Comments to the Author(s)
The manuscript has been improved accordingly, and I don't have any further questions.

Reviewer: 2 Comments to the Author(s)
In the manuscript resubmitted by Lu et al., the authors characterized the phenotypic and transcriptomic changes in the ARK1 overexpression lines compared to wild type Populous lines.

Decision letter (RSOS-201201.R1)
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. seedlings. How many lines did choose to maintain from qPCR data? Answer: We found that the expression levels of eight transgenic '717' lines and ten transgenic '84K' lines were found to be at least twice more than that of the control seedlings. What are the reference genes? And so on. Answer: We used the EF1 β as the reference gene.  The gene expression of AHP, CYCD3, and PER27 revealed by RT-qPCR analysis was basically the same as that of transcriptome analysis, and there was no significant difference (see Figure 6C). 6. The manuscript is not well written enough scientifically and grammarly. The authors did not introduce zeatin, brassinosteroids, phenylpropanoid pathways, either branching studies. Answer: We have discussed the relationships between the branching and zeatin, brassinosteroids and phenylpropanoid pathways in the Discussion part, although we did not present them in the Introduction part. Because we found that zeatin, brassinosteroids and phenylpropanoid pathways are related to branching after we analysis the transcriptome data, we thought it is not proper to present it in the Introduction part. And in the discussion, some parts are not very relevant to this manuscript. Answer: We have deleted the parts that is not very relevant to this manuscript. "The main synthetic site of zeatin and brassinosteroids is in the root, and they will enter the stem and leaf with the transportation of water. When the content in the stem increases, it will lead to the active bud in the stem." Lacking reference for some statements is also an issue in this manuscript. Answer: We have added some references in the manuscript. Many thanks for the letter. We have revised the manuscript according the the editor and reviewer's comment. Because we are not English native speaker, there are may be some places that we did not express very well. If there any thing we do in the wrong way, please let us know, we will try our best to revise it.
Associate Editor Comments to Author (Dr James Locke): The reviewers appreciate your efforts on the revision. However, one reviewer still has issues with some of the claims and the justifications of the manuscript. I propose that in a further revision you downplay some of your claims as the reviewer suggests and justify further your choice of focus (as well as moving the plasmid map etc into supplementary). Answer: Thanks. We have revised the manuscript according to the reviewer's comment. We have try to downplay some claims. For example, we have deleted the sentence "ARK1 is an important growth-related gene" from the conclusion section, and we have changed "its gene network regulation is poorly investigated." into "transcriptomic responses of enhancing-expression of ARK1 gene in poplar are poorly investigated." Also, we have move the Figure 1 and Figure 2 into supplementary.
Additionally, the RNAseq data should be made publicly available before publication. There is currently no result after searching PRJNA633952 in NCBI, which is the reference given for the dataset. Answer: We have made the RNAseq data be publicly available.
Reviewer comments to Author: Comments to the Author(s) The manuscript has been improved accordingly, and I don't have any further questions.

Reviewer: 2
Comments to the Author(s) In the manuscript resubmitted by Lu et al., the authors characterized the phenotypic and transcriptomic changes in the ARK1 overexpression lines compared to wild type Populous lines. The authors improved this manuscript, but there are still some important issues that need to be addressed: Major Comments: 1. The quality of the current manuscript is still not good enough. Even though, probably the authors tried to make a small story, it is need to be a qualified story. We do not get why the authors decided to study this ARK1 gene. The authors said that "The ARBORKNOX1 (ARK1) gene is an important gene for regulating plant growth and development; however, its gene network regulation is poorly investigated." There are many genes are important for plant growth. Moreover, the authors did not really In this study, to analyze the function of the ARK1 gene, the poplar lines '717' and '84K' were subjected to gene transform, the morphology and transcriptome profiles were compared between transgenic lines and control plants, and the expression levels of some genes were verified by RT-qPCR. The reviewer said that the authors did not really studied the network regulation. So we changed "its gene network regulation is poorly investigated." into "transcriptomic responses of enhancing-expression of ARK1 gene in poplar are poorly investigated." There are genes either up-or down-regulated in the transgenic lines from RNA-seq data.
However, any (if not, most genes should be the case) gene is over-expressed in plant, would cause transcriptomic changes.
Answer: Although any gene is over-expressed in plant, would cause transcriptomic changes. The expression levels of specific genes changed is unique to ARK1 over-expressed. The authors did not study the hierarchical relationship between ARK1 and transcription factors, associated proteins, and their target genes. There are transcriptomic changes in the transgenic plants. But, how does ARK1 affect them? Answer: Liu et al. (2014) used chromatin co-immunoprecipitation sequencing (chip-seq) to determine the binding sites of ARK1 in the poplar genome and found that ARK1 binds to thousands of sites, which are highly enriched in the transcriptional initiation sites of genes with different functions. We thought that Liu et al. (2014) should have described the target genes in their study. We are sorry that we don't know how to study the the hierarchical relationship between ARK1 and transcription factors, associated proteins, and their target genes. Maybe using the chromatin co-immunoprecipitation sequencing we could do more research on the relationships between ARK1 and other transcription factors, associated proteins, and their target genes, and on how ARK1 affect the transcriptomic changes. We did not described the how does ARK1 affect the transcriptomic changes in the manuscript. Meanwhile, we have described the transcriptomic changes in the transgenic plants.
2. There are thousands of DEGs in the ARK1 overexpressed lines and there are many pathways or hormones affect branching phenotype in nature. It is still not clear for us why did the authors choose zeatin, brassinosteroids, phenylpropanoid pathways to give a deeper transcriptomic description.
Answer: The stem segment of transgenic poplar '717' and '84K' had multiple branches. Zeatin and brassinosteroids were known to be involved in the branching phenotype, and phenylpropanoid pathway was found to be related to plant development, in our knowledge. Hence, it could be possible to find the reason why the ARK-transgenic plant have multiple branches.
Although there are thousands of DEGs in the ARK1 overexpressed lines and there are many pathways or hormones affect branching phenotype in nature. We did not find DEGs with padj < 0.05 and abs(log2FoldChange) > 1 in other pathways or hormones affect branching phenotype. Most important, we did not find any other DEGs annotated in Gene Ontology which had been previously reported to be involved in the branching phenotype.