A Type A Response Regulator Is Involved in Growth in Salix Matsudana Koidz

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear author, please find my comments and queries on the attached ms pdf file. 

Comments for author File: Comments.pdf

Author Response

All were corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

Overall the authors presented an interesting study regarding the identification of a growth promoting gene in willow. Although the science is sound, the presentation of the work in the manscript should be improved.

Introduction

The introduction should be more indepth. A possible expansion could include explaning currently known roles of RRAs and RRBs and their expression during general plant growth and development before going into the direct impacts of the knockout mutants.  A better case could be made for why it is an important gene to examine in willow.

Methods

For expression analysis I'm assuming the PCR assay was qPCR?

A clear rationale should be given as to only why 10 Arabidopsis type-A RR sequences were compared to SmRR5 and other willow homologs should be included.

Results

Please include units in the axis of Figure 1. Not all figures are of the same quality and resolution. Some figures have small writing that cannot be read when zooming in and thus a higher resolution figure should be provide (i.e. Figure 5).

Figure 2, which shows the sequences of SmRR5, should have conserved motifs indicative of a type-A RR highlighted. SmRR5 from YJ should also be sequenced to confirm that differences in expression are not due to structural differences. Sequencing of upstream and downstream segments may provide additional information as to why there are differences in gene expression.

Type A response regulator genes are induced by external application of cytokinin. This inducibility should have been tested in both willow cultivars. They are also considered to be activated by a class of MYB transcription factors. This information should be included in the introduction and the corresponding sequence highlighted in figure 2 if the sequence is available.

In Figure 3 it is unclear as to why only 10 Arabidopsis genes were chosen and no additional Salix genes are included.

Quantification of SmRR5 overexpression in each line should be completed. This would support why L2 was chosen for transcriptomics.

Discussion
The discussion does not do the study justice and link the transcriptomic changes to possible modes of function for SmRR5. The data from the previous transcriptome analysis between the two willow cultivars should be referenced here to support the data and results of this study.

Comments on the Quality of English Language

With regards to the writing itself, it should be read over as articles are occasionally missing (should be placed before a noun). 

More should be done to link the information presented in the paragraphs within both the introduction and discussion. 

Author Response

The introduction should be more in depth. A possible expansion could include explaning currently known roles of RRAs and RRBs and their expression during general plant growth and development before going into the direct impacts of the knockout mutants.  A better case could be made for why it is an important gene to examine in willow.
Reply：The introduction section was modified.

For expression analysis I'm assuming the PCR assay was qPCR?
Reply：Yes

A clear rationale should be given as to only why 10 Arabidopsis type-A RR sequences were compared to SmRR5 and other willow homologs should be included.
Reply: In the Arabidopsis genome, ARR genes were mainly classified into two groups. SmRR5 is homologous to 10 genes in group I. Thus, we just conducted the sequence alignment among them to see the bases changes. And, we did not select other willow genes since this research here just focused on SmRR5.

Please include units in the axis of Figure 1. Not all figures are of the same quality and resolution. Some figures have small writing that cannot be read when zooming in and thus a higher resolution figure should be provide (i.e. Figure 5).

Reply: Some modifications were made in figure 1.

Figure 2, which shows the sequences of SmRR5, should have conserved motifs indicative of a type-A RR highlighted. SmRR5 from YJ should also be sequenced to confirm that differences in expression are not due to structural differences. Sequencing of upstream and downstream segments may provide additional information as to why there are differences in gene expression.

Reply: Some modifications were made in figure 2. We had cloned the gene from YJ, which are same as that from 9901. We also cloned their promotors, in which several SNPs were found. We will start the expression project in next to find the possible regulators and see their difference in two varieties in binding and expression level.

Type A response regulator genes are induced by external application of cytokinin. This inducibility should have been tested in both willow cultivars. They are also considered to be activated by a class of MYB transcription factors. This information should be included in the introduction and the corresponding sequence highlighted in figure 2 if the sequence is available.
Reply: We did not test the external application of cytokinin on willow since we want to see the real performance of SmRR5 in tissues and organs of two varieties that will help us understand its contribution. We agree that exploring its regulator is necessary and will start in next.

In Figure 3 it is unclear as to why only 10 Arabidopsis genes were chosen and no additional Salix genes are included.

Reply: In the Arabidopsis genome, ARR genes were mainly classified into two groups. SmRR5 is homologous to 10 genes in group I. Thus, we just conducted the sequence alignment among them to see the bases changes. And, we did not select other willow genes since this research here just focused on SmRR5.

Quantification of SmRR5 overexpression in each line should be completed. This would support why L2 was chosen for transcriptomics.

Reply: We had quantified the expression level of SmRR5 in each line, which showed similar. L2 was selected since more clonal were obtained.

The discussion does not do the study justice and link the transcriptomic changes to possible modes of function for SmRR5. The data from the previous transcriptome analysis between the two willow cultivars should be referenced here to support the data and results of this study.

Reply: Modifications were made in discussion section.

With regards to the writing itself, it should be read over as articles are occasionally missing (should be placed before a noun). 

Reply: Some corrections were made.

More should be done to link the information presented in the paragraphs within both the introduction and discussion. 

Reply: It was done

Reviewer 3 Report

Comments and Suggestions for Authors

Studying the processes of plant growth regulation is one of the most important tasks. The study of the growth processes of woody plants is significantly complicated due to the extended period of their growth. To prove the participation of the SmRRs genes in growth stimulation, the authors transformed it into a fast-growing plant - tobacco.

33 - DNA abbreviation - generally accepted, you don’t have to decipher it

38 - in situ - in italic

page 3 - empty

77, 78 - remove underlining

98 - 10 mg/L space

Figure 6 - genes in italics

221 - RR genes are not gene-specific, but specific genes in regulation

format references according to the requirements of the magazine, uniformly

Author Response

33 - DNA abbreviation - generally accepted, you don’t have to decipher it

Reply: It was deleted.

38 - in situ - in italic

Reply: It was corrected

page 3 – empty

Reply: It was deleted

77, 78 - remove underlining

Reply: It was done

98 - 10 mg/L space

Reply: It was corrected

Figure 6 - genes in italics

Reply: All were corrected

221 - RR genes are not gene-specific, but specific genes in regulation

Reply: It was corrected

format references according to the requirements of the magazine, uniformly

Reply: All were checked

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The introduction could use a better description of what traits mentioned with regards to Arabidopsis would be useful in willow. For example biomes where the willow has been planted, difficulties growing in those areas, what is needed to be able to expand the range and suitability of planting willow in China. A strong link to how this research relates to forests.

Figure 1c need units in the y axis. The resolution of Figure 5ab needs to be improved to be legible.

Other than those minor edits, the text has been improved and is suitable for publication.

Author Response

The introduction could use a better description of what traits mentioned with regards to Arabidopsis would be useful in willow. For example biomes where the willow has been planted, difficulties growing in those areas, what is needed to be able to expand the range and suitability of planting willow in China. A strong link to how this research relates to forests.

Reply: Several references were added. Some modifications were made.

Figure 1c need units in the y axis. The resolution of Figure 5ab needs to be improved to be legible.

 Reply: All were corrected

Other than those minor edits, the text has been improved and is suitable for publication.

Reply: We go through the manuscript and revised