FvMYB44, a Strawberry R2R3-MYB Transcription Factor, Improved Salt and Cold Stress Tolerance in Transgenic Arabidopsis
Round 1
Reviewer 1 Report
Peer review for „R2R3-MYB transcription factor FvMYB44 of Fragaria vesca is involved in the salt and cold tolerance in Arabidopsis thaliana“ by Li et al.
The authors' work has an interesting focus, and the data show that the transcription factor FvMYB44 may play a significant role in strawberry resistance to abiotic stress.
On the other hand, the design of the experiment and the overall processing of the article makes it impossible to accept it. Firstly, the article is generally written in very unreadable English, which greatly detracts from the information provided. Many sentences have no verb, and some have no meaning (eg line 201: "In young leaves."). In this regard, I recommend sending the article to professional language proofreaders.
The main problem with the article is its inconsistency, unclear methodology and results.
Inconsistency:
line 90 – treatment by 100 µM ABA vs. Line 213 – 50 µM ABA
line 187 – the transformation of tobacco vs. Image 3 – localization in onion
Image 3b – „GFP“ and „Bright“ cannot result in a red signal in „Merge“
Line 227 – image 5c should show survival, but graph 5C is showing the germination rate
Line 88 – high temperature treatment 37 °C vs. Line 212 heat = 30 °C
Line 108 – Agrobacterium injected into tobacco vs. Line line 195 – transformation by particle bombardment
Line 155 and 157 – „…..FvMYB44 had a full length of 1, 221 bp……. FvMYB44 protein was composed of 99 aa“ vs. Line 295 „FvMYB44 had a 1 bp nucleotide sequence that encoded 410 aa (Figure 1)“ moreover figure 1 is showing 185 aa
Unclear methodology and results:
Line 84 Why did authors use a high concentration of cytokinins and auxins for control conditions is not clear. Moreover, mock treatment was not done.
Since some information in the methodology is not the same as in the results – we actually cannot be sure what is shown in the result part.
Why did the authors choose selected genes for analysis of homology? Moreover, Shukla et al. (10.1093/aobpla/plv054) already showed the homology of FvMYB44 with MdMYB. Why did the authors didn´t include this gene in their analysis? Why is this previous work not discussed?
Issues in discussion:
Discussion is mainly a recap of the results; it is not discussion per se. It should be more focused on comparing new data with already known information (E.g., the function of related AtMYB44 is known for a long time)
A not clear idea on line 307 – The higher expression of FvMYB44 in young leaves cannot be explained by higher sensitivity to stress, because these plants were not exposed to stress according to methodology. This idea is not clear to me.
A not clear idea – line 299 – FvMYB44 is similar to PaMYB73-like indicating that FvMYB44 regulates responses like RcMYB???
Some other issues:
Line 105 - word „protein“ is probably wrong in the context of digesting by endonuclease
Line 121 – concentration of primers has to be provided – volume is not sufficient information
Figures 6 and 8 – labelling cannot be read
The title should be changed – the title evokes that Fragaria gene FvMYB44 is actually present in Arabidopsis species
Multiple problems with the formatting of the text
Summary:
On the basis of the results in Figures 5, 6, 7, and 8, I believe that authors could present interesting data about FvMYB44, but in the presented version I see too many reasons why the paper should be significantly rebuilt. I would like to encourage authors to remake the article, in the sense that readers will fully understand the experiments, will be not confused by conflicting information and the data will be properly discussed.
Author Response
Thank you for your patience, we added the response below.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Manuscript review "R2R3-MYB transcription factor FvMYB44 of Fragaria vesca is involved in the salt and cold tolerance in Arabidopsis thaliana" by Wenhui Li et al.
MYB is widely involved in the response process of plant environmental factors. Most MYB proteins play the role of transcription factors. All data indicated that FvMYB44 may is an important candidate gene for improving salt stress and cold stress tolerance of strawberry.
Questions:
L.89-91. What is the duration of stress treatment?
L. 109. What were tobacco plants used for?
L. 138 For Columbia ecotype A. thaliana, the temperature is -8 C for 14 hours - there is a very strong cold stress. Why was this cooling mode chosen?
L. 143 The authors write that "Determine the absorbance of the extracted chlorophyll solution according to the method of Wang [35]". However, the link {35} does not contain this method that Wang developed. In [35], the authors used the method of Sartory and Grobbelaar (1984). The misunderstanding needs to be corrected.
L. 146 The manufacturer of the MDA analysis kit (TBA method) must be specified.
L. 211-213 The authors write that "Time-course of FvMYB44 expression in the control and under treatment with salt (200 mM NaCl), heat (30 °C), cold (4 °C), dehydration (15% PEG6000) , and abscisic acid (50μM ABA)." However, these experiments are missing in the Methods section.
L. 205 The authors write “….its peak at 4h, 2h, 4h, 6h, and 6 h,…..”. There seems to be a mistake here!
L. 228. The authors write "(L1, L3, and L5)." However, in fig. 5с missing L5. See the legend for the figure. Fix it.
Figure 6a and 8a - Change the legend label to "chlorophyll".
Figure 6 is hard to see - you need to increase the size.
L. 241 The authors write "(L1, L3, and L5)." However, in fig. 6 missing L5. See the legend for the figure. Fix it.
L. 242-243 The authors write "Significant differences were marked with asterisks above the error bar (** P ≤0.01)." Why didn't the authors use ANOVA?
“Discussion” resembles a retelling of the data obtained by the authors. There is little discussion of literature data.
Author Response
Thank you for your patience, we add the response below.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear authors, thank you for the improvement of the manuscript. I was originally against accepting the article, but after the appropriate adjustment, I am inclined to accept it. Since there are many grammatical mistakes in the article, I highly recommend professional language proofreading. More comments are below.
Key:
Line 82 Authors should be aware that CKs are used for seed/seedling priming to increase stress resistance (examples 10.1007/s10725-022-00861-6; 10.3389/fpls.2022.1103088). Moreover, CKs are not necessary for strawberry cultivation (10.1186/s12870-021-03095-2; 10.1016/j.bbrc.2021.06.085, 10.3390/ijms231911961). The authors must include an explanation for CK and auxin treatment and the whole manuscript must reflect the potential role of these hormones in priming and impact on the results, or experiments without hormonal treatment should be presented.
Line 141 – Authors analyzed data by ANOVA and Tukey´s test for multiple comparisons – authors should show these results in graphs by standard visualization with significantly different groups
Other issues:
Line 23 unclear sentence „However, for malondialdehyde (MDA), ROS O2- and H2O2）it is higher in WT and UL lines.“
Line 47 italics should be used for Zea mays
Line 47-48 unclear sentence
Line 50 missing dot
Line 51 What is meant by seedling rate of seeds?
Line 59 not a clear sentence: „The genetic transformation system of F. vesca is stable, it can be used as the dominant subgenome of cultivated strawberry. “
Line 73 two verbs: „may is“
Line 83 it is not clear what experiments were done in the soil and on the media
Line 90 probably a mistake in PEG – as the following text suggests the 6000 PEG was used
Line 92 and the following Time scales do not fit; results for stem are not shown
Line 99-100 " first-strand cDNA might be created“ sentence is not clear - should be explained in more detail
Line 101 "was selected"
Line 121 sentence requires correction
Line 122 missing verb
Line 126 sentence requires correction
Line 139 What is the unloaded line?
Line 142 missing a verb and I suggest using term “line” instead of “strain” as it is in other parts of the text
Line 151 sentence requires correction
Line 153 sentence requires correction
Line 155 sentence requires correction
Line159 There is a missing name of the test used in the text – according to previous part it is Tukey´s test
Line 170 Authors probably meant species, not varieties
Line 172 sentence requires correction
Line 187-189 Used reference has to be cited
Line 196 figure 2 is figure 3
Line 197 not ubiquitous – (e.g. not in chloroplasts)
Line 199 sentence requires correction
Line 206 term RT-qPCR is recommended
Line 207-209: The sentence is not clear and numbers do not fit (young leaves have the almost same expression as roots in Fig4a). I also recommend using the term young and old or developed leaf, instead of juvenile leaf with a different meaning.
208 typo „pof“
209 typo “In young leaves. The expression…”
224 I recommend “condition” instead of “settings”
231 Relative to what?
Fig 5 Is it known why 10 % of all lines are dying in control conditions?
Fig 5 Recovered plants don´t look healthy – did plants survive a longer period (more than 1 week)? Survival assay was not described.
Fig.7 It seems there is a position effect – two pots show 0 % surviving plants other three pots show 100 % survival. Did the authors check the homogeneity of the drought conditions? Our Percival growth chamber has position effects because of different air flows and we were forced to perform drought experiments very carefully.
284- control condition is 25°C according to line 88 – what is correct?
Line 327 missing reference
323 and following - genes should be in italics
335 Idea that young leaves are more susceptible to external stimuli should be supported by reference
345 sentence is not clear - it requires correction
352 not a clear sentence “Pressure will broke the metabolic balance of chlorophyll…”
365 Reformulate the first sentence
Author Response
Thank you for your patience and suggestion, we revised it. Please see the attachment.
Best regards.
Deguo Han
Author Response File: 
Author Response.doc
