PhSPEAR1 Participates in Regulating the Branch Development of Petunia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to evaluate the work. Here are some considerations.

Introduction:

In the last paragraph of the introduction, the connection with the previous paragraph could be improved.

Methodology:

Which program was used to create and validate the primer? Add it to the methodology.

The design of the qRT-PCR experiment could be improved for better understanding.

Results:

“The full length of the PhSPEAR1 gene was 579 bp, encoding a protein of 192 amino acids. It was speculated that the molecular formula of the PhSPEAR1 protein was C896H1417N273O309S8, the relative molecular weight was 21.2 KD, the isoelectric point was 8.54, and the instability parameter was 69.80. The highest content of PhSPEAR1 was Ser (19.8%, 38), and the lowest content was Trp (0.05%, 1) and Cys (1.0%, 2). Asp + Glu (total negatively charged residues) had a total of 18, and Arg + Lys (total positively charged residues) had a total of 20. It was speculated that the theoretical half-life was 30h.” Very cool information, where did you get it from? Put this in Methodology.

 

“Figure 1. Evolutionary tree analysis and protein structure analysis of SPEARs. NLS, SPL-motif, and EAR motif are all marked by black lines.”

Suggestion: Comparison of SPEARs proteins. (A) Evolutionary maximum-likelihood tree analysis; (B) Protein structure analysis of SPEARs. NLS, SPL-motif, and EAR motif are all marked by black lines.

 

“Figure 2. Expression analysis of PhSPEAR1 in different tissues of petunia.”

Indicate that the model used was Petunia × hybrid cv. Mitchell Diploid.

 

"Figure 3. Expression analysis of PhSPEAR1 under different treatments."

 

Indicate in the legend what each acronym of the treatments used means.

 

"Figure 5: (A) Comparison of phenotypes of WT and transgenic Arabidopsis plants after 30 d of growth; (B) Comparison of phenotypes of WT and transgenic Arabidopsis plants after 30 d of growth"

Check the legends of Figure 5 (A) and (B), they are the same.

 

Add what the acronyms stand for. I believe the acronym OE stands for Over Expression, and WT for Wild Type, but people who don't work with gene expression may read your work and get confused.

 

I couldn't find any explanation of what 6-BA is in the text. I had to look it up to understand that it is 6-benzylaminopurine, a synthetic cytokinin. Please add this information to the text.

 

174 - 176: "The results suggest that petunia SPEAR and Arabidopsis SPEAR1 are closely related (Figure 1A), suggesting that the petunia SPEAR gene may be a homologue of Arabidopsis SPEAR1, hence the name PhSPEAR1."

 

Why did they deduce that it was a homologue of SPEAR1 and not SPEAR3, since both groups together with PhSPEAR1? Develop this explanation.

 

References:

 

Species name is not italicized:

Yin, P.; Ma, Q.; Wang, H.; Feng, D.; Wang, X.; Pei, Y.; Wen, J.; Tadege, M.; Niu, L.; Lin, H. SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula. New Phytol. 2020, 226, 1399-1412. DOI: 10.1111/nph.16449.

 

Clough, S.J.; Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735-743. DOI: 10.1046/j.1365-313x.1998.00343.x

 

Sun, D.; Zhang, L.; Yu, Q.; Zhang, J.; Li, P.; Zhang, Y.; Xing, X.; Ding, L.; Fang, W.; Chen, F.; Song, A. Integrated Signals of Jasmonates, Sugars, Cytokinins and Auxin Influence the Initial Growth of the Second Buds of Chrysanthemum after Decapitation. Biology (Basel) 2021, 10, 440. DOI: 10.3390/biology10050440

 

Author Response

Introduction:

In the last paragraph of the introduction, the connection with the previous paragraph could be improved.

We improved it.

Methodology:

Which program was used to create and validate the primer? Add it to the methodology.

We have added it.

The design of the qRT-PCR experiment could be improved for better understanding.

We improved it.

Results:

“The full length of the PhSPEAR1 gene was 579 bp, encoding a protein of 192 amino acids. It was speculated that the molecular formula of the PhSPEAR1 protein was C896H1417N273O309S8, the relative molecular weight was 21.2 KD, the isoelectric point was 8.54, and the instability parameter was 69.80. The highest content of PhSPEAR1 was Ser (19.8%, 38), and the lowest content was Trp (0.05%, 1) and Cys (1.0%, 2). Asp + Glu (total negatively charged residues) had a total of 18, and Arg + Lys (total positively charged residues) had a total of 20. It was speculated that the theoretical half-life was 30h.” Very cool information, where did you get it from? Put this in Methodology.

 We have added it.

“Figure 1. Evolutionary tree analysis and protein structure analysis of SPEARs. NLS, SPL-motif, and EAR motif are all marked by black lines.”

Suggestion: Comparison of SPEARs proteins. (A) Evolutionary maximum-likelihood tree analysis; (B) Protein structure analysis of SPEARs. NLS, SPL-motif, and EAR motif are all marked by black lines.

 Very good suggestion, and we are very happy to accept it.

“Figure 2. Expression analysis of PhSPEAR1 in different tissues of petunia.”

Indicate that the model used was Petunia × hybrid cv. Mitchell Diploid.

 We have revised it.

"Figure 3. Expression analysis of PhSPEAR1 under different treatments."

Indicate in the legend what each acronym of the treatments used means.

  We have added it.

"Figure 5: (A) Comparison of phenotypes of WT and transgenic Arabidopsis plants after 30 d of growth; (B) Comparison of phenotypes of WT and transgenic Arabidopsis plants after 30 d of growth"

Check the legends of Figure 5 (A) and (B), they are the same.

 We have revised it.

Add what the acronyms stand for. I believe the acronym OE stands for Over Expression, and WT for Wild Type, but people who don't work with gene expression may read your work and get confused.

 We have added it.

I couldn't find any explanation of what 6-BA is in the text. I had to look it up to understand that it is 6-benzylaminopurine, a synthetic cytokinin. Please add this information to the text.

 We have added it.

174-176: "The results suggest that petunia SPEAR and Arabidopsis SPEAR1 are closely related (Figure 1A), suggesting that the petunia SPEAR gene may be a homologue of Arabidopsis SPEAR1, hence the name PhSPEAR1."

Why did they deduce that it was a homologue of SPEAR1 and not SPEAR3, since both groups together with PhSPEAR1? Develop this explanation.

We have revised it. The sequence similarity between SPEAR and Arabidopsis SPEAR1 is higher, so we modified it to combine sequence alignment.

References:

Species name is not italicized:

Yin, P.; Ma, Q.; Wang, H.; Feng, D.; Wang, X.; Pei, Y.; Wen, J.; Tadege, M.; Niu, L.; Lin, H. SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula. New Phytol. 2020, 226, 1399-1412. DOI: 10.1111/nph.16449.

Clough, S.J.; Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735-743. DOI: 10.1046/j.1365-313x.1998.00343.x

Sun, D.; Zhang, L.; Yu, Q.; Zhang, J.; Li, P.; Zhang, Y.; Xing, X.; Ding, L.; Fang, W.; Chen, F.; Song, A. Integrated Signals of Jasmonates, Sugars, Cytokinins and Auxin Influence the Initial Growth of the Second Buds of Chrysanthemum after Decapitation. Biology (Basel) 2021, 10, 440. DOI: 10.3390/biology10050440

We carefully revised the references.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Information needs to be attached and homogenized.

If possible, the authors should indicate at what phenological stage the samples were taken in the experiments; they mention the age, but not the phenology.

What standards were used for the detection of hormones, and how they determined the presence of these hormones.

Homogenize, the age of the plants in days and not in weeks of development

The figures that present bars, it is necessary to describe what statistical test they applied

In Figure 3, it is suggested to explain why the bar in decapitation treatment is so high.

The discussion, if it is possible to write it again, in many of the paragraphs what was written in results is being repeated; the conclusion is not capitulated.

Although the citations are included, I suggest that it be described how the result is related to what was obtained with the authors of the citations.

It would be important to indicate what influence the age of the plants has on the results obtaine

It is suggested to point out the structures and increases in figure 4

Author Response

Information needs to be attached and homogenized.

If possible, the authors should indicate at what phenological stage the samples were taken in the experiments; they mention the age, but not the phenology.

We are appreciate for the reviewer's suggestions. I don't know how phenology is represented. Can you briefly tell us how to modify it?

What standards were used for the detection of hormones, and how they determined the presence of these hormones.

We usually test all hormones except SLs and then check which hormones have changed. Because the vast majority of companies are unable to accurately detect the content of SLs.

Homogenize, the age of the plants in days and not in weeks of development

We have revised it.

The figures that present bars, it is necessary to describe what statistical test they applied

We have revised it.

In Figure 3, it is suggested to explain why the bar in decapitation treatment is so high.

We plan to redo it because the bar is indeed very high and we don't know how to explain it.

The discussion, if it is possible to write it again, in many of the paragraphs what was written in results is being repeated; the conclusion is not capitulated

Although the citations are included, I suggest that it be described how the result is related to what was obtained with the authors of the citations.

We have rewritten the discussion section.

It would be important to indicate what influence the age of the plants has on the results obtaine

It is suggested to point out the structures and increases in figure 4

We don't quite understand the meaning here? we hope the reviewer can provide more detailed suggestions.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors evaluated the expression and function of PhSPEAR1 using qPCR and overexpression analyses. They also analyzed the localization of the PhSPEAR1 protein. 

While I feel confident in the experimental quality, the manuscript and data presentation needs to be fixed. More edits should be made to help their readers understand why they took the steps they took.

The M&M need to be heavily revised. It seems as though several authors wrote the M&M, and it needs to be edited so that only one of the author's voices can be heard when reading the text. Many key details are missing and need to be added. The order of M&M could be rearranged for better clarity, too. Maybe regrouping it by the experiment(s) conducted? Having one author go through and put everything into one voice would be the most helpful thing.

I'm unsure why the authors used petunias at different ages for their experiments unless I missed it; the text did not explain it. Again, this goes back to the poorly written M&M.

The other question I have is about the number of SPEAR genes. There was no discussion of other potential SPEAR homologs in Petunia. They listed several species with multiple SPEAR genes. It seems strange that Petunia would only have one. While I feel it is fine to just publish a paper on one gene, I find it odd that they did not mention any others out there.

The authors' presentation of the results can also be improved. Below is a summary outline that should help fix the results/discussion sections.

1) First, we identified potential homologs and examined their divergence and secondary protein structures from potential homologs in Arabidopsis.

2) The one ortholog from petunia shared key structures, so we wanted to see if it was upregulated when branch-inducing treatments were applied. We will measure these changes through qPCR.

3) As a gene regulator, it is likely localized in the nucleus, which has been demonstrated in other species. Using a GPF fusion protein, we found that it was localized to the nucleus. Therefore, it likely interacts with genetic material.

4) While the candidate gene in Petunia was divergent from Arabidopsis, key protein structures of the SPEAR family were maintained. Therefore, we tested the gene's function by overexpressing the PhSPEAR gene in Arabidopsis to see if it would induce branching, which it did. We also examined the canonical hormonal shifts that occur after decapitation. The same shifts were also observed in the hormone levels collected from the plants.

These data combined demonstrate that we have identified a true homolog of SPEAR in petunia.

 

Other very important comments need to be addressed prior to publication in the uploaded file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

The M&M need to be heavily revised. It seems as though several authors wrote the M&M, and it needs to be edited so that only one of the author's voices can be heard when reading the text. Many key details are missing and need to be added. The order of M&M could be rearranged for better clarity, too. Maybe regrouping it by the experiment(s) conducted? Having one author go through and put everything into one voice would be the most helpful thing.

We have revised it.

I'm unsure why the authors used petunias at different ages for their experiments unless I missed it; the text did not explain it. Again, this goes back to the poorly written M&M.

In the experiment of  tissue expression, we originally detected the expression levels of PhSPEAR1 in the roots, stems, leaves, axillary buds, and flowers of petunia. So we need flowering plants. However, we tested four times in total and found that the expression level in flowers were not consistent, so we removed the flower tissue.

In the treatment experiment, we needed plants with ungerminated axillary buds, so we used 40-d-old petunia.

The other question I have is about the number of SPEAR genes. There was no discussion of other potential SPEAR homologs in Petunia. They listed several species with multiple SPEAR genes. It seems strange that Petunia would only have one. While I feel it is fine to just publish a paper on one gene, I find it odd that they did not mention any others out there.

In fact, we have cloned two SPEAR genes, and the other gene is still under further research, so we did not mention the other gene in this article.

The authors' presentation of the results can also be improved. Below is a summary outline that should help fix the results/discussion sections.

1) First, we identified potential homologs and examined their divergence and secondary protein structures from potential homologs in Arabidopsis.

2) The one ortholog from petunia shared key structures, so we wanted to see if it was upregulated when branch-inducing treatments were applied. We will measure these changes through qPCR.

3) As a gene regulator, it is likely localized in the nucleus, which has been demonstrated in other species. Using a GPF fusion protein, we found that it was localized to the nucleus. Therefore, it likely interacts with genetic material.

4) While the candidate gene in Petunia was divergent from Arabidopsis, key protein structures of the SPEAR family were maintained. Therefore, we tested the gene's function by overexpressing the PhSPEAR gene in Arabidopsis to see if it would induce branching, which it did. We also examined the canonical hormonal shifts that occur after decapitation. The same shifts were also observed in the hormone levels collected from the plants.

These data combined demonstrate that we have identified a true homolog of SPEAR in petunia.

We have rewritten the discussion, which may not be good enough.

Other very important comments need to be addressed prior to publication in the uploaded file.

the topic of the manuscript is very interesting but many changes are needed in all sections: in the introduction is necessary to better explain the reason for which the gene SPEAR was chosen for analysis; in the material and methods it is not clear the number of plants used, in the results the statistical analysis must be improved, also using the ANOVA test. Finally, it's not clear why to study the role of this gene in  petunia, subcellular localization was carried out in tobacco and the overexpression in Arabidopsis. In the attached file more suggestions are reported

We have made revisions based on the comments provided by the reviewers in the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors

the topic of the manuscript is very interesting but many changes are needed in all sections: in the introduction is necessary to better explain the reason for which the gene SPEAR was chosen for analysis; in the material and methods it is not clear the number of plants used, in the results the statistical analysis must be improved, also using the ANOVA test. Finally, it's not clear why to study the role of this gene in  petunia, subcellular localization was carried out in tobacco and the overexpression in Arabidopsis. In the attached file more suggestions are reported

Comments for author File: Comments.pdf

Author Response

the topic of the manuscript is very interesting but many changes are needed in all sections: in the introduction is necessary to better explain the reason for which the gene SPEAR was chosen for analysis; in the material and methods it is not clear the number of plants used, in the results the statistical analysis must be improved, also using the ANOVA test. Finally, it's not clear why to study the role of this gene in  petunia, subcellular localization was carried out in tobacco and the overexpression in Arabidopsis. In the attached file more suggestions are reported

Much appreciated for the reviewer's detailed and professional advise. We have made detailed modifications according to the advise in the attachment. There may still be some imperfections, please let us know. There are a few other issues that we would like to clarify.

We use pSuper1300 GFP for subcellular localization, not pCAMBIA1300;

Nicotiana benthamiana is a commonly used subcellular localization material, and tobacco is grown by ourselves.

75-day-old tobacco plants are flowering, and we can detect gene expression in flowers. This plant age cannot be used for bud treatment experiment because many axillary buds have already sprouted at this time. Generally, 40-day-old plants are used. At this point, the axillary buds are just right to be treated.

The basal axillary buds of a 75-day-old petunia plants have sprouted, but the top axillary buds have not yet sprouted.

Author Response File: Author Response.docx