Genome-Wide Analysis of Poplar SQUAMOSA-Promoter-Binding Protein (SBP) Family under Salt Stress

Round 1

Reviewer 1 Report

The research group have successfully studied phylogeny, gene structure, cis-acting elements, chromosome distribution and gene duplication of a total of 30 SBP genes from poplar. The research is conducted in organized and transparent manner. The manuscript is well written and organized too. But there are some topics has repetitive presence. I would suggest 10-15% of reduction of the introduction part. Please correct spelling errors (minor). The labels of the figures are mostly unreadable. Use larger fonts and use larger space for figures. Otherwise the manuscript is good to be published in present form.

Author Response

Dear reviewers,

Thank you for your letter and the comments concerning our manuscript entitled

“Genome-wide analysis of poplar SQUAMOSA-Promoter-Binding Protein (SBP) family under salt stress" (ID: forests-1118187). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. The main corrections in the paper and the responds to the reviewer's comments are as flowing:

1. Response to comment:

I would suggest 10-15% of reduction of the introduction part. Please correct spelling errors (minor).

 Response: 

Thank you for your valuable advice. I have reduced the introduction by 14%(delete “AtSPL8 was expressed in pistil and can regulate the growth of female genital tract plant and pollen sac. In addition,” in line 47-48. delete “In grape, as many as 11 SBP genes were induced in the long photoperiod, indicating that the expression of SBP genes was regulated by photoperiod. In addition, ” in line 56-57) and corrected spelling errors(“cing-acting” be converted into “cis-acting” in line 74) .

2. Response to comment:

The labels of the figures are mostly unreadable. Use larger fonts and use larger space for figures.

 Response: 

Thank you for your valuable advice. I have used larger fonts and larger space for figures.

Kind regards,

Guoqing

Reviewer 2 Report

Colleagues,

I read your manuscript and came to the conclusion that you have done a lot of painstaking work. I see that your work consists of two parts: in silico studies of genes and experiments on living plants. I think that the information obtained by you in the experiments is much more valuable than the computer analysis of the data that were not obtained by you. Better to put more emphasis on experimentation. They have novelty and practical interest. Unfortunately, I do not see any valuable information in the results of your computer analysis. I ask you to explain to me the novelty, value and practical significance of the results in parts 3.2, 3.3, 3.4. Maybe these parts are better to reduce or eliminate altogether?

About part 3.3. What is the purpose of localization of studied genes on chromosomes? What species of poplar do chromosomes belong to?

Generally, you write "poplar" throughout the article and do not specify what Populus species you mean. This is a very gross mistake. I urge you to correct the text in all places where you write "poplar". Concretize! In the genome of which poplar species did you search for genes? Why did you take the Populus simonii × Populus nigra hybrid for experiments? Why didn't you use any species of poplar? It would be better to use the same species as in computer analysis. I ask you to give detailed answers to these questions.

Small comments:

Line 71-72. The phrase “few studies have been studied” must be worded differently.

Fig. 1,3,5,6,7 are not clear. Please, increase their quality.

Author Response

Dear reviewers,

Thank you for your letter and the comments concerning our manuscript entitled

“Genome-wide analysis of poplar SQUAMOSA-Promoter-Binding Protein (SBP) family under salt stress" (ID: forests-1118187). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. The main corrections in the paper and the responds to the reviewer's comments are as flowing:

1. Response to comment:

In the genome of which poplar species did you search for genes? Why did you take the Populus simonii × Populus nigra hybrid for experiments? Why didn't you use any species of poplar?

 Response: 

Thank you for your careful work. The experimental object of this study is Populus simonii×P.nigra. Compared with other poplar varieties, Populus simonii×P.nigra has specific features such as fast growth, strong adaptability and cold resistance, which can grow in alpine areas such as Heilongjiang, China. It has been proved by years of cultivation and management. However, the Populus simonii×P.nigra genome has not been published. Populus trichocarpa is woody model plant and typical representative species for the forest genetic study. Its genome has been measured and published in 2006, which is relatively mature and effective. We conducted a large number of RNA-Seq for Populus simonii×P.nigra, with founding that it had similar gene sequences with Populus trichocarpa were high homology, whose blast rate could reach more than 99%. Therefore, the genome of Populus trichocarpa was used as the reference genome in this study.

2. Response to comment:

I ask you to explain to me the novelty, value and practical significance of the results in parts 3.2.

 Response: 

Thank you for your careful work. The valuable content of chapter 3.2 is that phylogenetic tree analysis has great instruction significance in studying the diversity of gene family and its function. Phylogenetic tree reveals the evolutionary relationship of homologous SBP gene and clustering relations of AtSBPs, OsSBPs and PtSBPs. It also help predict the functionality of homologous PtSBPs according to proven Arabidopsis thaliana and Oryza sativa SBP genes research. Gene structure variation reflects the unity and diversity of gene family structure. All genes in this study contain the SBP domain, confirming they are members of the SBP family. PtSBP4, PtSBP14, PtSBP17 and PtSBP27 contain both SBP domain and Ank_2 domain which are all distributed in Group V. They possibly have the same new specific function. This study provide reference for subsequent study.

3. Response to comment:

I ask you to explain to me the novelty, value and practical significance of the results in parts 3.3. About part 3.3. What is the purpose of localization of studied genes on chromosomes? What species of poplar do chromosomes belong to?

Response: 

Thank you for your careful work. In chapter 3.3, genes mapping in chromosome is helpful to study those distribution and further analyze the evolution of PtSBPs, with reference to the chromosomes and genomes of Populus trichocarpa. The expansion of any species gene family is mainly implemented by periodic replication events. This study analyzes PtSBPs’ duplicate genes to explore the origins of PtSBPs and reveal their evolutionary relationships. Gene duplication is the driving force for reproduction of new genes and expansion of gene family, which provides the original genetic resources for neutral selection and organisms adoption under different complex environments. Ka/Ks is considered to be an authoritative boundary value for determining gene purification selection or neutral selection. Statistics show that the Ka/Ks replacement rate below, equal to or higher than 1 indicates purification, neutral and positive selection, respectively. We calculated Ka/Ks substitution rate of gene duplication pairs. All Ka/Ks values are far less than 1, indicating that poplar SBP gene family has experienced strong purification selection, which removed harmful mutations to maintain function possibly. Therefore, the biological function of poplar SBP genes can be inferred by comparing with orthologous genes form Arabidopsis. There are four SBP homologous pairs found, including PtSBP3-AtSBP11, PtSBP13-AtSBP6, PtSBP24-AtSBP10, PtSBP27-AtSBP12. The functions of AtSBP11, AtSBP6, AtSBP10 and AtSBP12 have been verified, so we speculate that the corresponding PtSBPs may have similar functions. This study provides a useful reference to future research of PtSBPs and its function.

4. Response to comment:

I ask you to explain to me the novelty, value and practical significance of the results in parts 3.4.

 Response: 

Thank you for your careful work. In chapter 3.4, Cis-acting elements play an important role in gene transcriptional regulation during plant development and in the response to stress. In this study, we found that there are a large number of elements involving growth and development, abiotic stress, stress resistance and hormone response. So we infer PtSBPs may participate in the important physiological and developmental processes of plants, responding to the external environment. Among them, the promoters of PtSBP12 and PtSBP13 contain lots of abiotic stress and hormone response elements, whose subsequent experiments also show that they respond to salt stess significantly. MiRNA participates in the gene regulation at post-transcriptional stage in plants, and plays an important role in growth and development and stress responses. A large amount of PtSBPs have target relationships with miR156, miR164, miR167 and miR7826. It can be inferred that these genes may have the same function as targeting miRNA. For example, many studies have shown that miR156 can play a role under drought, salt, cold and heat stress. 14 PtSBPs have target relationships with miR156, all distributed in groups IV and VI. It is indicate that they may have similar functions. PtSBP13 was confirmed to respond significantly to salt stress in subsequent experiments. This study provides a useful reference to future research of PtSBPs and its function.

5. Response to comment:

Generally, you write "poplar" throughout the article and do not specify what Populus species you mean. This is a very gross mistake. I urge you to correct the text in all places where you write "poplar".

Response: 

Thank you for your valuable advice. I have corrected the text. Line 13, 94, 201, 237, 238, 433, 438, 526. The phrase “poplar” be converted into “Populus trichocarpa”. Line 80. The phrase “poplar” be converted into “Populus simonii×P.nigra”.

6. Response to comment:

Line 71-72. The phrase “few studies have been studied” must be worded differently.

Response: 

Thank you for your careful work. Line 71-72. The phrase “few studies have been studied” be converted into “few studies have been conducted on”.

7. Response to comment:

Fig. 1,3,5,6,7 are not clear. Please, increase their quality.

 Response: 

Thank you for your valuable advice. I have increase the quality of Fig. 1,3,5,6,7.

Kind regards,

Guoqing

Round 2

Reviewer 2 Report

Thank you for your responses.