Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study discusses the role of microtubules in shaping the morphogenesis of Salix psammophila plants under various environmental conditions. It focuses on the expression patterns and genetic structures of tubulin genes, specifically SpsTUB10, in response to different stresses.

Overall, this study provides valuable data on the role of microtubules in different plant types of S. psammophila. However, the manuscript should be checked for grammar and punctuation errors in the English language.

My specific comments are below:

-In line 148, Salix purpurea L. needs to be italicized. In line 536 Salix psammophila must be replaced with S. psammophila. The name of the Sps-tubulin genes must be mentioned in the whole manuscript in the same format. It is written in different formats as in lines 537, 547, 451,..

In line 483, SpsTUB10 needs to be written in nonitalic format. Tubulin genes are italicized in the whole manuscript except the title, please correct it. In figure 3, the scientific names of the different species need to be italicized.

- In the "Materials and Methods" section, it is better to add a subtitle titled "Plant Materials" and explain the accepted methods for different ions of three plant types: Salix psammophila. It is also important to mention the names of the clones in the first sentence.

- In lines 133-135, RNA-seq was performed on different tissues from 27 clone samples. However, it is unclear if this number of samples is accurate, as the previous lines mentioned only three clone samples from three plant types of S. psammophila. Therefore, the number of samples should be nine, not 27. Could you please confirm this? Additionally, what is the scientific rationale for choosing the terminal bud, fresh stem, and root for gene expression analysis? How many replications were conducted for RNA-seq? It would be helpful to provide more information on the materials and methods used in this study.

- What is the reason for selecting just SpsTUB10 to examine the relative expression between different tissues (based on line 170) and also for further analysis? Why are different plants (one-year-old) and different tissues chosen for total RNA extraction compared to the sampling mentioned in section 2.1?

-In Fig. 3, the predicted amino acid tertiary structure of SpsTUB10, doesn't make sense and can be removed.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Revises and Explanations

Dear Reviewers and Editors:

Thank you for your letter and for the reviewer’s comments concerning our manuscript entitled “Evolutionary characterization of tubulin gene family in the desert biomass willow (Salix psammophila) and expression of the β-tubulin gene SpsTUB10 during different stresses” (MS ID: forests-2923331). Those comments are all valuable and helpful for improving and revising our manuscript. We have studied comments seriously and made correction. Apart from the questions raised by the reviewers, we carefully checked the entire text and corrected several expressions, symbols, and grammar errors. The changes were marked in the original manuscript using the track-changes feature of Microsoft Word. The lists of our answers for the reviewers’ comments as follows.

Thank you again for your time and effort in handling our manuscript.

Reviewer 1

Quality of English Language

( ) I am not qualified to assess the quality of English in this paper

( ) English very difficult to understand/incomprehensible

( ) Extensive editing of English language required

( ) Moderate editing of English language required

(x) Minor editing of English language required

( ) English language fine. No issues detected

Yes Can be improved Must be improved Not applicable

Does the introduction provide sufficient background and include all relevant references?

(x) ( ) ( ) ( )

Are all the cited references relevant to the research?

(x) ( ) ( ) ( )

Is the research design appropriate?

( ) (x) ( ) ( )

Are the methods adequately described?

( ) (x) ( ) ( )

Are the results clearly presented?

( ) (x) ( ) ( )

Are the conclusions supported by the results?

(x) ( ) ( ) ( )

Comments and Suggestions for Authors

The study discusses the role of microtubules in shaping the morphogenesis of Salix psammophila plants under various environmental conditions. It focuses on the expression patterns and genetic structures of tubulin genes, specifically SpsTUB10, in response to different stresses.

Overall, this study provides valuable data on the role of microtubules in different plant types of S. psammophila. However, the manuscript should be checked for grammar and punctuation errors in the English language.

Reply: We are highly grateful for your positive contents. We have checked the MS carefully to revise English language and grammatical editing of the manuscript.

My specific comments are below:

In line 148, Salix purpurea L. needs to be italicized. In line 536 Salix psammophila must be replaced with S. psammophila. The name of the Spstubulin genes must be mentioned in the whole manuscript in the same format. It is written in different formats as in lines 537, 547, 451,..

Reply: Thanks for your valuable advice. Done across the MS.

In line 483, SpsTUB10 needs to be written in nonitalic format. Tubulin genes are italicized in the whole manuscript except the title, please correct it. In figure 3, the scientific names of the different species need to be italicized.

Reply: Done.

In the "Materials and Methods" section, it is better to add a subtitle titled "Plant Materials" and explain the accepted methods for different ions of three plant types: Salix psammophila. It is also important to mention the names of the clones in the first sentence.

Reply: Done. In lines of 128-141 of the 2.1 section.

In lines 133-135, RNA-seq was performed on different tissues from 27 clone samples. However, it is unclear if this number of samples is accurate, as the previous lines mentioned only three clone samples from three plant types of S. psammophila. Therefore, the number of samples should be nine, not 27. Could you please confirm this? Additionally, what is the scientific rationale for choosing the terminal bud, fresh stem, and root for gene expression analysis? How many replications were conducted for RNA-seq? It would be helpful to provide more information on the materials and methods used in this study.

Reply: The total number of transcriptome samples is 27, with three replications for each of the three tissues for each of the three plant types. This can be confirmed in the Figure 1D. We have revised the related contents to avoid confusion. The scientific rationale for choosing tissues for RNA-seq has been illustrated and you can find it in 2.2 Plant tissue collection and RNA sequencing.

What is the reason for selecting just SpsTUB10 to examine the relative expression between different tissues (based on line 170) and also for further analysis? Why are different plants (one-year-old) and different tissues chosen for total RNA extraction compared to the sampling mentioned in section 2.1?

Reply: Among these 26 SpsTubulins, ten members (green box for each of them, Figure 1D) have the full lengths in CDS sequences compared with their homologous genes, including SpsTUA1, 2, 6, 7, and SpsTUB3, 6, 7, 9, 10, and 20 (Figures S1 and S4). Selecting genes of these ten SpsTubulines will promise the consistence between the assembled SpsTubulins using RNA-seq and the cloned ones via PCR and sanger sequencing. Previous study showed that high sequence similarity, evolutionary expansion and expression pattern diversity of Salix β-tubulin gene families might confer flexibility in cell division and growth which is of important significance to the development and growth habit of perennial woody plants (Sui et al., 2016). Moreover, the homologous gene of the SpsTUB10 showed a significantly different expression among different tissues within Salix (Sui et al., 2016). In particular, the SpsTUB10 shows a significantly higher expression level in the root and stem than in the bud in comparisons of the three plant types, with stable replications for each tissue. Such selection strategy might be beneficial in exploring the key SpsTubulins in S. psammophila according to development and environmental cues. Of course, the functional roles of more SpsTubulins will be explored both in the development and environmental demands in our future works. We have illustrated these points across the MS.

In Fig. 3, the predicted amino acid tertiary structure of SpsTUB10, doesn't make sense and can be removed.

Reply: The significant conservation in sequences roots in constraints imposed by the constitution of the tertiary structure of heterodimers of the tubulins. The consistent structures from secondary to tertiary further confirm the conservation within species (Figure 3E,F). The significant conservation in sequences across species can be achieved via interspecific comparisons, as shown in Figure. 3G. We thus used the secondary and tertiary structures within Salix psammophila to further document such conservation and thus it makes sense.

Comments on the Quality of English Language

Minor editing of English language required.

Reply: We have checked the MS carefully to revise English language and grammatical editing of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewer has a few minor comments:

1. In the abstract:

Under different 33 stresses, we found cold, drought and long-day light…

Long day conditions are not a stress factor in themselves. If they are such specifically for your object, this must be somehow justified in the article. If they are not, you should not consider the long day as a stress factor.

2. Title of Figure 3: What is shown in the figure is not the evolutionary characteristics of the gene, but rather its structure

3. Figure 4: It’s not very clear how A and C, B and D differ.

Author Response

Revises and Explanations

Dear Reviewers and Editors:

Thank you again for your time and effort in handling our manuscript.

Reviewer 2

Quality of English Language

( ) I am not qualified to assess the quality of English in this paper

( ) English very difficult to understand/incomprehensible

( ) Extensive editing of English language required

( ) Moderate editing of English language required

( ) Minor editing of English language required

(x) English language fine. No issues detected

Yes Can be improved Must be improved Not applicable

Does the introduction provide sufficient background and include all relevant references?

(x) ( ) ( ) ( )

Are all the cited references relevant to the research?

(x) ( ) ( ) ( )

Is the research design appropriate?

(x) ( ) ( ) ( )

Are the methods adequately described?

(x) ( ) ( ) ( )

Are the results clearly presented?

(x) ( ) ( ) ( )

Are the conclusions supported by the results?

(x) ( ) ( ) ( )

Comments and Suggestions for Authors

The reviewer has a few minor comments:

Reply: We are highly grateful for your positive contents. We have checked the MS carefully to revise the issues concerning long-day light, the title of Figure 3 and some contents in Figure 4.

In the abstract:

Under different stresses, we found cold, drought and long-day light…

Reply: In our MS, long-day light was set with the parameter of 15 h light and 9 h dark, which is shorter than the control (16 h) in light, we thus further classified the light-related stress treatments: all dark (24 h dark), shorter-day light (9 h light and 15 h dark), short-day light (15 h light and 9 h dark), all light (24 h light) , and control (16 h light and 8 h dark). We have revised the related contents across the MS. Toward this way, long-day light was not involved in and removed in the revised version of the MS. In addition, we defined the treatments as stress factors if they differ from the controls which usually are the best growing conditions for examining plants. We have illustrated this in the MS.

Title of Figure 3: What is shown in the figure is not the evolutionary characteristics of the gene, but rather its structure

Reply: Thanks for your valuable advice. We have checked that and revised as “Structure features of the SpsTUB10 gene in Salix psammophila.”

Figure 4: It’s not very clear how A and C, B and D differ.

Reply: All samples used to investigate the relative expression levels of the SpsTUB10 were hydroponically treated 24 h in a greenhouse (24℃, 16 h light and 8 h dark, located in Inner Mongolia Agricultural University) to make them as consistent as possible, especially the initial expression levels of the SpsTUB10 before stress treatments. For A and C, B and D, we did not make comparisons in morphology, but only focused on the changes of the SpsTUB10 transcript levels. Before temperature-related stresses treated, the SpsTUB10 transcript levels were nearly equal, without significance difference, as Figure 4E showed. This is also same in the other stress treatments (Figure 4J,L). We have further described this scenario in our MS and you can find it in 3.4. expression of the SpsTUB10 gene under various abiotic stresses.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

A fairly typical work for the present time has been completed. Two features of this manuscript I would like to point out. Firstly, the authors study a family of tubulin genes, which are rather conservative and because of their stability are often used as reference genes. However, the results show that genes of this family are differentially expressed in different organs and in response to external influences. The second feature is that the woody plant is studied. It is certainly not yet sufficiently studied and many methodological difficulties are encountered when working with this object.

By applying numerous bioinformatic approaches, which are well developed nowadays, the tubulin gene family is characterised. The analysis of the physical-chemical properties of proteins encoded by the SpsTubulin genes was carried out. Conserved domains characteristic for these proteins were revealed. The exon-intron structure for several genes was studied. A phylogenetic analysis based on amino acid sequences of the tubulin genes among different species was performed. Transcriptome analysis and RT-qPCR were used to study gene expression under salinity, osmotic stress, low and high temperature conditions.

This article provides new information on a poorly studied gene family in woody plants. The article is useful.

However, there are comments to which I would like to see a response from the authors. The line numbers to which comments are made are given on the left.

260: This paper did not study drought, per se, but PEG-induced osmotic stress, one of the negative consequences of which can be water deficit. The authors cannot even speak of water deficit from the available data, as it too must be characterised. Since "drought" is one small element of the paper, I suggest where it says “drought” replace it with “osmotic stress”.

258-261: How the plant treatment temperature of 4 ℃ and 40 ℃, PEG concentration (20% 260 PEG 6000) and 250 mM NaCl were determined. Usually, such parameters are determined in preliminary experiments or taken from the literature in case of well-studied plants such as A. thaliana.

The response of a plant to any factor depends on the plant species and its resistance. Many genes are now known which are markers that the plant is under stress under the action of low positive temperature, extreme high temperature, salt exposure and so on. It would be very useful to use such marker genes to check the condition of plants under the action of negative factors.

294-300: It is very correct that the authors confirmed gene expression using RT-qPCR, which was obtained by transcriptome analysis, but usually not one but several genes with different regulation character are used for this purpose. This is all the more important as the expression level of SpsTUB10 matched the transcriptome data in the root but not in the stem.

84: The authors see no distinction between stressor and stress. Stressor is an acting factor (like temperature, salinity, etc.) and stress is the state of the plant under the conditions of the stressor, i.e. the plant's response to the stressor.

95, 131, 133, 170, caption to Fig. 1 and elsewhere: The authors almost always refer to organs as tissues, and as we know, an organ is a more complex concept and almost always consists of several or many tissues.

179: 1.0 μL qSpsTUB10-F, 1.0 μL q-SpsTUB10-R. The volume does not tell anything, it is better to specify the molarity of the primer solution, then everything will be clear (same line 217).

186: UBQ (glycine soja ubiquitin) It is necessary to specify the accession numbers of this gene

Author Response

Revises and Explanations

Dear Reviewers and Editors:

Thank you again for your time and effort in handling our manuscript.

Reviewer 3

Quality of English Language

(x) I am not qualified to assess the quality of English in this paper

( ) English very difficult to understand/incomprehensible

( ) Extensive editing of English language required

( ) Moderate editing of English language required

( ) Minor editing of English language required

( ) English language fine. No issues detected

Yes Can be improved Must be improved Not applicable

Does the introduction provide sufficient background and include all relevant references?

(x) ( ) ( ) ( )

Are all the cited references relevant to the research?

(x) ( ) ( ) ( )

Is the research design appropriate?

(x) ( ) ( ) ( )

Are the methods adequately described?

( ) (x) ( ) ( )

Are the results clearly presented?

( ) (x) ( ) ( )

Are the conclusions supported by the results?

(x) ( ) ( ) ( )

Comments and Suggestions for Authors

Dear Authors,

Reply: We are highly grateful for your positive contents. We have checked the MS carefully to revise methods adequately and results of the manuscript.

By applying numerous bioinformatic approaches, which are well developed nowadays, the tubulin gene family is characterised. The analysis of the physical-chemical properties of proteins encoded by the SpsTubulin genes was carried out. Conserved domains characteristic for these proteins were revealed. The exon-intron structure for several genes was studied. A phylogenetic analysis based on amino acid sequences of the tubulin genes among different species was performed. Transcriptome analysis and RT-qPCR were used to study gene expression under salinity, osmotic stress, low and high temperature conditions.

This article provides new information on a poorly studied gene family in woody plants. The article is useful.

Reply: Thank you for your high evaluation.

However, there are comments to which I would like to see a response from the authors. The line numbers to which comments are made are given on the left.

260: This paper did not study drought, per se, but PEG-induced osmotic stress, one of the negative consequences of which can be water deficit. The authors cannot even speak of water deficit from the available data, as it too must be characterised. Since "drought" is one small element of the paper, I suggest where it says “drought” replace it with “osmotic stress”.

Reply: Done across the MS. Thanks for your valuable advice.

258-261: How the plant treatment temperature of 4 ℃ and 40 ℃, PEG concentration (20% 260 PEG 6000) and 250 mM NaCl were determined. Usually, such parameters are determined in preliminary experiments or taken from the literature in case of well-studied plants such as A. thaliana.

Reply: Done. In lines of 290-293 of the 2.6 section. Stress treatment sources were 4 °C and 42 °C, PEG concentration ( 22 % PEG 6000 ) ( Jia et al., 2019 ) and 250 mM NaCl ( Temel et al., 2015 ). We cited these stress treatments across the MS. On this basis, we passed the limits of stress treatment on Salix psammophila and the limits of possible stress treatment on SpsTUB10. Some adjustments were made to the stress conditions, the high temperature was reduced to 40 °C, and the PEG was reduced to 20 %.

The response of a plant to any factor depends on the plant species and its resistance. Many genes are now known which are markers that the plant is under stress under the action of low positive temperature, extreme high temperature, salt exposure and so on. It would be very useful to use such marker genes to check the condition of plants under the action of negative factors.

Reply: Thank you for your high evaluation.

294-300: It is very correct that the authors confirmed gene expression using RT-qPCR, which was obtained by transcriptome analysis, but usually not one but several genes with different regulation character are used for this purpose. This is all the more important as the expression level of SpsTUB10 matched the transcriptome data in the root but not in the stem.

Reply: Done. We have studied result seriously and made correction. In lines of 330-342 of the 3.1 section. In our MS, an extremely significant difference in the expression levels of the SpsTUB10 in the root and stem-developing organs (including axillary bud, stem, phloem and xylem) (P < 0.001), no significant difference of the SpsTUB10 expression was detected in the terminal bud (P > 0.05) (Figure 1E). The expression levels of the SpsTUB10 in the root and stem were consistent with the RNA-seq data, while that in the bud did not agree with the RNA-seq data (Figure 1D, E). We have illustrated these points across the MS.

84: The authors see no distinction between stressor and stress. Stressor is an acting factor (like temperature, salinity, etc.) and stress is the state of the plant under the conditions of the stressor, i.e. the plant's response to the stressor.

Reply: Done. We reviewed the relevant literature, agree with your point of view, has been modified. In lines of 93-96 of the Introduction section.

95, 131, 133, 170, caption to Fig. 1 and elsewhere: The authors almost always refer to organs as tissues, and as we know, an organ is a more complex concept and almost always consists of several or many tissues.

Reply: Done across the MS. Thanks for your valuable advice.

179: 1.0 μL qSpsTUB10-F, 1.0 μL q-SpsTUB10-R. The volume does not tell anything, it is better to specify the molarity of the primer solution, then everything will be clear (same line 217).

Reply: Done. We have added the concentration of these primers. In lines of 204-205 of the 2.4 section, and 243-244 of the 2.5 section.

186: UBQ (glycine soja ubiquitin) It is necessary to specify the accession numbers of this gene

Reply: Done. We have added the Genebank number of the UBQ. In lines of 212-213 of the 2.4 section.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewer's remarks have been taken into consideration, and the revised text of the MS may be accepted for publication.

Review Reports

In line 483, SpsTUB10 needs to be written in nonitalic format. Tubulin genes are italicized in the whole manuscript except the title, please correct it. In figure 3, the scientific names of the different species need to be italicized.