Characterization of CBL-Interacting Protein Kinases’ Gene Family and Expression Pattern Reveal Their Important Roles in Response to Salt Stress in Poplar

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Dear Authers,

I would say thank you for responding to my comments.

I think the revised manuscript would be reach to enough level to publish on Forestry.

Reviewer 2 Report (Previous Reviewer 3)

I believe that the authors have addressed the issues needed and it is acceptable. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors has done a great job no more comments 

Reviewer 2 Report

This manuscript describes Poplar's CIPK genes from an objective perspective and provided valuable scientific information.  I think that there is insufficient discussion of the results presented in the manuscript, and some figures would be improved.

 

Major comments

This manuscript show that the PtrCIPKs can be classified into two categories, few intron classes and multi-intron classes based on the genomic structure. You only discuss about this point that "gene structure of CIPK family was highly similar" among the plant species.

However, there is insufficient explanation on how they are similar. It is desirable to consider more deeply about the biological significance of the existence of these two types of genetic structure, such as what is known in other preceding organisms and what can be clarified from this result.

This manuscript show the arrangement of cis-elements in the promoter region of PtrCIPK in Fig 3, and the transcriptional levels in the salt stress treatment. But it seems that the discussion between these two results has not been touched upon. I seem both results as if they are dissociated.

In addition, some protein kinases respond to environmental stimuli at the transcriptional level, but in other cases they respond by phosphorylation activity. What do you consider about this point?

 

Minor comments

Figure 1

panel a: The tree lines and protein names are not visible (especially those drawed in red).

panel d: The one letter designation of the amino acid is barely visible, but the protein names are difficult to identify.

 

Figure 2

panel a:  The gene locuses are not visible.

panel b: The scientific names shouled be italicized. It is preferable to widen the space between each panel a little more.

 

Figure 4

Please show appropriate figure legend.

Please discribe what each of the panels a, b, and c represents in figure regend.

Clarify what the alphabet above the bar indicates in figure regend. In addition,  the method of statistical analysis should be described in the method section.

 

Figure 5

Please show appropriate figure legend.

Clarify what the alphabet above the bar indicates in figure regend. In addition,  the method of statistical analysis should be described in the method section.

 

There is no period in the last minute of the abstract. (line 13)

In line 35, "3-" is superscript.

In line 39, "+" is superscript.

In line 231, "+" is superscript.

In line 243, "Table S2" is redundant. 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Review for Forests – 1788453

Title: Characterization of CBL-interacting protein kinases gene family and expression pattern reveal their important roles in response to salt stress in poplar

Summary: This paper used both experimental and bioinformatics data to identify CIPKs in poplar. The authors focus on the expression responses of CIPKs under salinity stress, as they have been proven to be important in the salt stress signal transduction pathway.

Comments:

1.     The introduction feels inadequate. There is much more information regarding the signal transduction pathways for salt stress, and the authors barely mention any of it. CIPKs are indeed important, but if they want to talk about why they are important, they also have to mention how CIPKs help with the signaling to create gene expression responses and not just how CIPKs can control certain transporters. Salt stress is much more complicated than just preventing ion contents from escalating.

2.     How were the vascular tissues (xylem and phloem) separated? Were these only with the trunk, or were these also with other tissues? Perhaps images of the plants, along with a summary figure for what was sampled could be included.

3.     The machine used for the qRT-PCR analysis should be stated as well as the reference gene used in this section. How many replicates, both technical and biological, were used? How was statistical significance computed?

4.     Page 3, Line 11 – “the text continues here” should probably be omitted?

5.     The gene names on the leaves of Figure 1A are too small to read. Additionally, it might be better to add the birch CIPKs in this comparison as well to have a better view of the phylogenetic relationships. Please expound more on the figure legends for Figure 1 to make it easier to follow.

6.     In page 5, lines 145 to 151, it may be fairer to say that the function of CIPKs are conserved between dicots but have diverged more in monocots. Please also make a mention of the number of CIPKs in the representative monocot you have selected as a comparison of how much divergence occurred. And again, please expound more on the Figure legends to make it easier to follow. The figure legends are too bare bone.

7.     For the 12 homologs with corn, are they conserved for any function? Are these also conserved with Arabidopsis and birch?

8.     For the prediction of cis-elements, why only focus on those which are stress-related? Can the authors also make a mention of how many may be related to differentiation? Why are there so many CIPKs? Are they only for stress? And how were the cis-elements curated for their specified function?

9.     Which were the redundant genes untested for qPCR? Why were they redundant? How are the authors certain that they were redundant?

10.  First, the authors did not even state which tissue the qRT-PCRs were performed on in Figure 4. The caption is again, extremely bare and inadequate to explain what the data being shown is. The authors have a very confusing classification of their results for the qRT-PCRs. CIPK genes shown in both 4A and 4B both have inconsistent expressions, which have up and down patterns. The “peak” in the middle the authors are describing for 4B is not even present there. The authors should revise this and actually look at their data first and try to interpret it with the physiological data in mind. Is there any wilting observed at the 6-hour mark? When does the plant begin to acclimate? These are the things the authors should be asking when interpreting gene expression, and they should not try to just describe the data in a half-baked manner.

11.  PtCIPK9, 11, 12, 14, 24 counts as FIVE genes, not four. The authors should again, actually look at the data and not write this carelessly. In addition, ALL of the genes have significant changes in expression according to their data. Why was the focus put on these five genes? What was special about them? The authors even state on their discussion that all of the CIPK genes significantly changed expression.

12.  The authors also immediately jump into conclusions regarding the function of the CIPK just based on increased expression. There are a number of other physiological phenomena happening during salt stress.

The authors tried to present the work as a brief study to determine the PtCIPK genes. While the bioinformatics analysis presented is informative, the qRT-PCRs, the data presentation, and the writing are inadequate and needs to be expounded a lot to give necessary information. Those things limit the value of the manuscript when it could have been very helpful information especially since Ca²⁺ signaling is an important component of stress response.

 

Minor corrections:

Page 1 Line 19 – significantly expressed should be significantly differentially expressed

Page 2 Lines 31 to 39 – Please state the organism where this info was taken from. It is important to identify it properly since the gene names are being stated, and the particular homolog the authors are talking about may be different from one organism to another.

There are various minor grammar and spelling errors that should be corrected.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear authers,

Thank you for your sincere response to my comments.

It evaluates that figures and figure legends have improved.

 

I look forward to further considerations and proofs for the following two points.

 

#1

In line 245-248;

“In 245 addition, the prediction of cis-acting elements of PtrCIPKs found that stress-related ele- 246 ments contain more binding sites for MYB transcription factors and WRKY transcription 247 factors. Under salt stress, the changes in the expression of PtrCIPKs may be regulated by 248 upstream MYB and WRKY transcription factors.”

 

These descriptions are general comments but not corresponding to your results. In case of CIPK3 and CIPK6, it seems to agree with your added description. But how about in case of CIPK9 etc. For example, CIPK9, CIPK14, and CIPK21 expressions induced by salt stress (Figure 4), but there is neither “W-box” nor “MYB” in its promoter (Figure 3).

It prefers to describe some original discussions for your experimental data.

 

#2

In Authors note; 

“This is a very meaningful question. When CIPK functions as a protein kinase, it usually needs to be phosphorylated by the upstream CBL protein to function. The effect of phosphorylation on the function of CIPK protein will be investigated in future experiments. “

It prefers to describe these discussions in the manuscript, not in confidential communication.

 

 

Author Response

 "Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors still need to work on the manuscript. They did not address all the comments made.

Add in which organism the mentioned CIPKs are in the second paragraph. 

The figure on the different plant parts sampled was supposed to be added as a supplementary, because at the end of the day, it's for the readers of the article. 

Gene names in Figure 1A and 1D are barely readable. If it is hard to read at 200% zoom, will this even be visible when printed? The same issue is there for Figure 2. 

Having a high amino acid similarity does not make a gene redundant. Again, what is the basis for saying it's redundant? And the authors also did not put which genes are redundant. What if these genes are actually important and the ones that they picked are the homologs that are silenced? They could check this by comparing expression of the genes to each other, not to each timepoint. 

T-test is for pairwise comparisons of statistical significance. How are the authors doing this when they are comparing five timepoints? 

The authors also did not even mention which tissue was used for the temporal qRT-PCR experiments done. This was explicitly asked in the prior review. Also, what is the reference sample for the tissue-based qRT-PCR? That needs mention in the methods. 

Page 1 Line 20: 3.5 times

Author Response

 "Please see the attachment."

Author Response File: Author Response.pdf