Populus trichocarpa PtHSFA4a Enhances Heat Tolerance by Regulating Expression of APX1 and HSPs
Round 1
Reviewer 1 Report
This is an interesting article that describes the role of heat shock TF in enhancing heat tolerance in woody plants. The experiments are well planned and the results are clearly presented. There are some minor сomments:
L. 24. Names of plant species should be italicized.
L. 139. “Table 1” should be corrected to “Table S1”.
It is desirable to move the Table 1 (list of primers) to Suppl. Materials.
Subsection 2.4. RT-qPCR conditions should be specified.
L. 265. “Eucalyptos” should be corrected to “Eucalyptus”.
L. 296. It should be noted that Arabidopsis transgenic seedlings were treated.
L. 386. The gene name should be italicized.
Author Response
Dear reviewer:
Thank you for your letter and comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We read the comments carefully and have made corrections which we hope to meet with approval. We decide to resubmit the manuscript and the revision were listed as following.
Major questions:
Comment 1: L. 24. Names of plant species should be italicized.
Our response: It was changed.
Comment 2: “Table 1” should be corrected to “Table S1”
Our response: “Table 1” was corrected to “Table S1”
Comment 3: Subsection 2.4. RT-qPCR conditions should be specified.
Our response: “The RT-qPCR conditions were pre-denaturation at 94 ℃ for 40 s, followed by 40 cycles of denaturation at 94 ℃for 10 s, an annealing and extension step for 1 minute at 60 ℃ and with a final extension step at 72 ℃ for 10 min.“ was added in Subsection 2.4.
Comment 4: L. 265. “Eucalyptos” should be corrected to “Eucalyptus”.
Our response: “Eucalyptos” was corrected to “Eucalyptus”.
Comment 5: L. 296. It should be noted that Arabidopsis transgenic seedlings were treated.
Our response: “GUS staining in 5-day-old ProPtHSFA4a:GUS transgenic seedlings treated with or without high temperature (45°C, 1 h). Scale bar = 200 µm. Three independent experiments were performed using at least 5–6 seedlings per experiment with similar results.” was changed to “GUS staining in 5-day-old ProPtHSFA4a:GUS transgenic Arabidopsis seedlings treated with or without high temperature (45°C, 1 h). Scale bar = 200 µm. Three independent experiments were performed using at least 5–6 Arabidopsis seedlings per experiment with similar results.”. Arabidopsis transgenic seedlings was noted.
Comment 6: L. 386. The gene name should be italicized.
Our response: “AtHSP101” was italicized.
Reviewer 2 Report
Comments and Suggestions for Authors
The present study aimed to gain a more in-depth and comprehensive understanding of the structure of the HSF family gene PtHSFA4a from Populus trichocarpa involved in response to heat stress of the plant. The importance of the study is defined by the needs of overcoming the affect of high ambient temperature on photosynthetic efficiency, cell division and growth, plant growth, development and fertility, and the necessity of genetic improvement of the adaptability of trees to cope with global warming trends in order to ease the heat threat. The thermotolerance mechanisms involving HSFs are well known in Arabidopsis thaliana, rice, and tomato. The available information regarding the transcriptional modulation of HSFs in woody species in response to heat stress is little. The expression of PtHSFA4a was performed by reverse transcription-quantitative PCR and GUS analyses. To investigate the biological function of PtHSFA4a, it was ectopically overexpressed in A. thaliana.
The manuscript is constructed according to requirements of “Forests”. The research methods applied are appropriate, comprehensive and sufficient to achieve the objectives of the study. The figures are representative and of good quality. The statistical analysis applied complement and support the results.
A continuation, some recommendations are given:
- The aim needs detailing. The passage that follows sounds like a result and should be edited to support the aim.
- - In "Materials and methods" it is indicated that Arabidopsis thaliana and Populus trichocarpa were used as a source of plant material, but the starting plant material (explant) for obtaining seedlings is not specified.
- - In the first paragraph of "Discussion", the text between lines 428-436 is more suitable for "Introduction" and duplicates the information presented in it. I suggest that this paragraph begin as follows: “The response of plants to high temperature is mainly regulated by HSFs. In this study, we identified an HSF-type …..”
- The references are not properly described in the list of references
According to “Instructions for Authors” of “Forests”, the literature sources have been described as follows:
· Journal Articles:
1. Author 1, A.B.; Author 2, C.D. Title of the a- this rticle. Abbreviated Journal Name Year, Volume, page range. - in the present manuscript the journal name are not in italic, the years - not in bold, and they are not in the rigt order
In conclusion, this manuscript is recommended for publication in “Forests”, after consideration of the remarks shown.
Author Response
Dear reviewer:
Thank you for your letter and comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We read the comments carefully and have made corrections which we hope to meet with approval. We decide to resubmit the manuscript and the revision were listed as following.
Major questions:
Comment 1: In "Materials and methods" it is indicated that Arabidopsis thaliana and Populus trichocarpa were used as a source of plant material, but the starting plant material (explant) for obtaining seedlings is not specified.
Our response: ”Seeds of Arabidopsis thaliana ecotype Columbia-0 were obtained from the Arabidopsis Biological Resources Center (ABRC) and Populus trichocarpa seedings clone Donglin plants.” was added in "Materials and methods" .
Comment 2: In the first paragraph of "Discussion", the text between lines 428-436 is more suitable for "Introduction" and duplicates the information presented in it. I suggest that this paragraph begin as follows: “The response of plants to high temperature is mainly regulated by HSFs. In this study, we identified an HSF-type …..”
Our response: “Greenhouse gas emissions have significantly altered the global climate, increasing the frequency and intensity of heat stress events, and are expected to continue to do so. ” and “However, the function and regulatory mechanism of HSFs in heat stress tolerance have mainly focused on herbaceous plants, whereas information on the role of woody plant HSF proteins in thermotolerance remains scarce at best, particularly with regard to the regulation of target genes.” were deleted, and “Here, we identified an HSF-type transcription factor” was changed to “In this study, we identified an HSF-type transcription factor”.
Comment 3: The references are not properly described in the list of references
Our response: The reference style followed in this journal.
Reviewer 3 Report
The manuscript entitled “Populus trichocarpa PtHSFA4a enhances heat tolerance by reg-2 ulating expression of APX1 and HSPs” demonstrated that PtHSFA4a responds to heat stress and acts as a positive regulator of heat tolerance by regulating antioxidant defense responses and maintaining protein-folding homeostasis in A. thaliana leaves. The results draw a possible molecular mechanism involving PtHSFA4a in heat stress responses and provide novel insights into the HSFA4a of woody plant species.
The manuscript is generally well-written and structured. The analysis was successful, and the data was well understood and modeled in detail. In addition, the manuscript contains relevant paragraphs that have been discussed. The selection of the bibliography is appropriate to the content of the manuscript.
However, some errors appeared throughout the manuscript, making it difficult to accept it in its current version.
- The authors must follow the reference style of the journal.
- Please be sure that all the genes’ names are in italics.
- It is highly recommended to move Table 1 to the supplemental material.
- The figure legend of Figure 1 can be summarized, especially in 1D; you can involve the scientific names in the tree itself. Moreover, indicate what it means the red color of PtHSFA4a.
- Cite a reference from lines 428 to 437.
Author Response
Dear reviewer:
Thank you for your letter and comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We read the comments carefully and have made corrections which we hope to meet with approval. We decide to resubmit the manuscript and the revision were listed as following.
Major questions:
Comment 1: The authors must follow the reference style of the journal.
Our response: The reference style followed in this journal.
Comment 2: Please be sure that all the genes’ names are in italics.
Our response: We check all the genes’ names in the manuscript and all the genes’ names were in italics.
Comment 3: It is highly recommended to move Table 1 to the supplemental material.
Our response: “Table 1” was corrected to “Table S1”.
Comment 4: The figure legend of Figure 1 can be summarized, especially in 1D; you can involve the scientific names in the tree itself. Moreover, indicate what it means the red color of PtHSFA4a.
Our response: The figure legend of Figure 1D was summarized and the scientific names were added after the gene name in the tree. The red color of PtHSFA4a highlights the PtHSFA4a genes that are the focus of this study.
Comment 5: Cite a reference from lines 428 to 437.
Our response: “Greenhouse gas emissions have significantly altered the global climate, increasing the frequency and intensity of heat stress events, and are expected to continue to do so. ” and “However, the function and regulatory mechanism of HSFs in heat stress tolerance have mainly focused on herbaceous plants, whereas information on the role of woody plant HSF proteins in thermotolerance remains scarce at best, particularly with regard to the regulation of target genes.” were deleted, and the retained content was added references.
Author Response File: 
Author Response.pdf
Reviewer 4 Report
The manuscript titled "Populus trichocarpa PtHSFA4a enhances heat tolerance by regulating expression of APX1 and HSPs " investigates the role of PtHSFA4a, a heat shock transcription factor, in regulating plant responses to heat stress in Populus trichocarpa and Arabidopsis thaliana.
The work is in the scope of the journal and given the importance of understanding stress tolerance mechanisms, the information provided might be of interest to a fair spectrum of readers. The text effectively presents the background, objectives, and results of the research, and the methods are in general well described, providing relevant information for its intended audience. The results are in general well presented, although I think it would be interesting to provide the fold change instead of just the relative expression.
Nonetheless, I have my concerns regarding the statistical analysis carried out. The authors say a “one-way analysis of variance (ANOVA)” was performed (Lines 227-229). However, in Figures 3, 4 and 6 captions, authors state: “Asterisks indicate significant differences at *P < 0.05 and **P < 0.01, as per Student’s t-test”. Which one was used, ANOVA or t-test? Furthermore, if the t-test was the one used, please indicate which specific comparisons were made in each figure. For example, I assume in Figure 4, the mean from each line (OE1, OE2 and OE3) were only compared to the mean from WT and not between then. Please specify in each figure what comparison was made.
Furthermore, I believe Figures 2, 4, 5 and 6 would benefit from a makeover, in order to have a more professional look. In the current state they are a bit crowded and kind of difficult to read.
Other specific comments
Lines 114-115: “16 h photoperiod” is enough, instead of “16 h light/8 h dark photoperiod”
Line 117: Was the medium supplemented with any carbon source, growth regulator, jellifying agent? What was the medium pH? Please add more information.
Line 122: Were the samples flash frozen in liquid nitrogen before stored at -80, or were stored directly at -80?
Lines 136-138: What model was used in the maximum likelihood method? Due to the reference in line 138, I assume it was Tamura model. However, authors should clearly state this, and why was this model selected. Did the authors used the “find best model” tool from MEGA? Other reason? Just to be sure, this was done with actually protein sequences, using this type of model?
Line 182: “Petri” instead of “petri”
Lines 204-205: “at least”?
Author Response
Dear reviewer:
Thank you for your letter and comments concerning our manuscript. These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We read the comments carefully and have made corrections which we hope to meet with approval. We decide to resubmit the manuscript and the revision were listed as following.
Major questions:
Comment 1: Nonetheless, I have my concerns regarding the statistical analysis carried out. The authors say a “one-way analysis of variance (ANOVA)” was performed (Lines 227-229). However, in Figures 3, 4 and 6 captions, authors state: “Asterisks indicate significant differences at *P < 0.05 and **P < 0.01, as per Student’s t-test”. Which one was used, ANOVA or t-test? Furthermore, if the t-test was the one used, please indicate which specific comparisons were made in each figure. For example, I assume in Figure 4, the mean from each line (OE1, OE2 and OE3) were only compared to the mean from WT and not between then. Please specify in each figure what comparison was made.
Our response: Figure 3 and Figure 4 were used Student’s t-test. Figure 6 was used one-way analysis of variance (ANOVA). In 2.10. statistical analysis, “The SPSS (version 18.0) software was used to perform one-way analysis of variance (ANOVA). In all the reported tests, single and double asterisks indicate P < 0.05 and P < 0.01, respectively” was corrected to “The SPSS software was used to perform statistical analyses. Statistically significant differences are evaluated using Student’s t test or one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) posthoc test. In Student’s t test, single and double asterisks indicate P < 0.05 and P < 0.01, respectively. In one-way ANOVA, different letters above the data points are used to indicate differences that are statistically significant".
In Figures 3 captions, “Asterisks indicate significant differences between WT and overexpression of PtHSFA4a lines: *P < 0.05, **P < 0.01, as per Student’s t-test.” was corrected to “Statistically significant differences between control condition and heat stress condition were analyzed by Student’s t-test (*P < 0.05, **P < 0.01)”.
In Figures 4 captions, “Asterisks indicate significant differences as determined using a Student’s t-test, *P < 0.05 and **P < 0.01” was corrected to “Statistically significant differences between WT and PtHSFA4a-overexpressing lines (OE1, OE2 or OE3) were analyzed by Student’s t-test: *P < 0.05 and **P < 0.01”.
In Figures 6 captions, “Asterisks indicate significant differences at *P < 0.05 and **P < 0.01, as per Student’s t-test” was corrected to “Different letters above the columns indicate significant differences (P < 0.05), as determined by one-way ANOVA”.
Comment 2: Furthermore, I believe Figures 2, 4, 5 and 6 would benefit from a makeover, in order to have a more professional look. In the current state they are a bit crowded and kind of difficult to read.
Our response: Figures 2, 4, 5 and 6 made over.
Comment 3: Lines 114-115: “16 h photoperiod” is enough, instead of “16 h light/8 h dark photoperiod”
Our response: “16 h light/8 h dark photoperiod” was corrected to “16 h photoperiod”.
Comment 4: Line 117: Was the medium supplemented with any carbon source, growth regulator, jellifying agent? What was the medium pH? Please add more information.
Our response: “3-week-old P. trichocarpa seedlings were cultured in vitro on 1/2 Murashige and Skoog (MS) medium supplemented with 6% (w/v) PEG6000, 150 mM NaCl, 25 mM ABA, 80 μM CdCl2, 100 μM CuSO4, or 1.0 mM FeSO4, as stress treatments, and incubated for 0, 1, 3, 6, and 12 h. As for heat stress, 3-week-old wild-type P. trichocarpa seedlings were incubated at 45°C in the dark for 0, 1, 3, 6, and 9 h” was corrected to “Additionally, 3-week-old P. trichocarpa seedlings were cultured in vitro on 1/2 Murashige and Skoog (MS) medium with 0.6% (w/v) agar, 2% (w/v) sucrose and respectively supplemented with 6% (w/v) PEG6000, 150 mM NaCl, 25 mM ABA, 80 μM CdCl2, 100 μM CuSO4, or 1.0 mM FeSO4, as stress treatments, and incubated for 0, 1, 3, 6, and 12 h. As for heat stress, 3-week-old wild-type P. trichocarpa seedlings were cultured in vitro on 1/2 MS medium with 0.6% (w/v) agar, 2% (w/v) sucrose and incubated at 45°C in the dark for 0, 1, 3, 6, and 9 h. The pH of medium set at 5.8-6.0” .
Comment 5: Line 122: Were the samples flash frozen in liquid nitrogen before stored at -80, or were stored directly at -80?
Our response: The samples flash frozen in liquid nitrogen before stored at -80℃.“Roots and leaves of P. trichocarpa exposed to various stress conditions cited above were sampled and stored in −80℃ refrigerator for gene expression analysis” was corrected to “Roots and leaves of P. trichocarpa exposed to various stress conditions cited above were sampled that were flash-frozen in liquid nitrogen and stored in −80℃ refrigerator for gene expression analysis”.
Comment 6: Lines 136-138: What model was used in the maximum likelihood method? Due to the reference in line 138, I assume it was Tamura model. However, authors should clearly state this, and why was this model selected. Did the authors used the “find best model” tool from MEGA? Other reason? Just to be sure, this was done with actually protein sequences, using this type of model?
Our response: We have to say sorry for the mistake and thank you for the reviewer’s question. Jones-Taylor-Thornton (JTT) model was used in the maximum likelihood method and the reference was deleted.
In 2.3. Phylogenetic Analysis of the HSFA4a Family, “Subsequently, a phylogenetic tree generated using the maximum likelihood method was visualized using the MEGA5.2 software.” instead of “Subsequently, a phylogenetic tree generated by maximum likelihood method in which distance was estimated by Jones-Taylor-Thornton (JTT) model implemented in the program MEGA5.2 software (https://www.megasoftware.net/download_form)”.
Comment 7: Line 182: “Petri” instead of “petri”.
Our response: “Petri” instead of “petri”
Comment 8: Lines 204-205: “at least”?
Our response: Some experiments were repeated four times.
Author Response File: Author Response.pdf
Round 2
Reviewer 4 Report
After the changes made by authors, i believe the manuscript is suitable to be published in Forests.
