Regulation of BcMYB44 on Anthocyanin Synthesis and Drought Tolerance in Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis Makino)
Round 1
Reviewer 1 Report
The authors have done great work related to the function of BcMYB44 in non-heading Chinese Cabbage and provide new insights into the roles of MYB44 in anthocyanin and drought stress. I have just some comments for improving the structure of the manuscript.
- Line 65: "Astragalus membranaceus" should be provided in italic form.
- In the title of Figures 8, 9, and 10, the components of the figure (A, B, etc.) should be defined.
- I suggest using this sentence in the INTRODUCTION or DISCUSSION section: Also, several potential interactions have been reported between flavonoid biosynthesis components and Sulfotransferase (SOT) proteins in potato (Faraji et al, 2021), and TIFY proteins in maize and tomato (Heidari et al, 2021).
Faraji et al, 2021: https://doi.org/10.3390/plants10122597
Heidari et al, 2021: https://doi.org/10.3389/fgene.2021.657970
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dear Authors and Editors,
In my opinion, the manuscript of the article is very well written. Authors widely explore the regulation of BcMYB44 on anthocyanin 15 synthesis and drought tolerance of non-heading Chinese cabbage. They showed convincingly in the manuscript, that BcMYB44 had a clear regulatory role in drought stress. Interestingly, the accumulation of anthocyanins is negatively correlated with resistance to drought stress.
A few small remarks:
Line 75-76. The main task should be more clearly defined,
Line 89-90. Drought treatment should be clarified and detailed - when was the investigation started, how long did it take, at which time samples were taken and what level of watering was for the control?
Line 148. How to explain sentence: “The ‘NJZX1-3’ and ‘NJZX1-0’ which were introduced into pTY empty plasmid (pTY-S) were used as control” Maybe it should be as “the plants ‘NJZX1-3’ and ‘NJZX1-0’ into which the pTY empty plasmid (pTY-S) was introduced, used as a control”?
Line 269. The reliability of the differences is not indicated in the figure 9.
Line 304. Instead “In addition to regulating movement” should be “In addition to regulating stomatal movement”.
Line 314-315. Figure 10. It should be explained that CT means control, DS - drought stress.
Line 326. Should be Liu at al, because are several authors in [33].
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript Regulation of BcMYB44 on Anthocyanin Synthesis and Drought Tolerance in Non-heading Chinese Cabbage is investigated an important relationship between drought and anthocyanin synthesis. The various experiments were carried out to clarify the role of transcription factor MYB44 in this process. A lot of work has been done, which, unfortunately, is not fully and qualitatively described in the manuscript. Some comments and suggestions are given below.
Abstract:
Lines 16-17: We cloned the sequence of BcMYB44 from ‘NJZX1-3’ and ‘NJZX1-0 . Better to clarify what are ‘NJZX1-3’ and ‘NJZX1-0.
Lines 19-20: Subcellular location – perhaps, better use the term localization?
Lines 21-22: Complimentary (BiFC) experiments showed that BcMYB44 interacted with BcPAP1/BcEGL3. What is BcPAP1/BcEGL3 ?
Line 25: VIGS further confirmed … No full name of this abbreviation.
Abstract is overwhelmed with protein short names, abbreviations and details of experiments. Perhaps, an abstract should be rewrite to make it shorter and more strongly clarify the main results and conclusion of your research.
Introduction
Line 35: the Brassica genus of the cruciferous family … Perhaps, it is more correct to use the latin name – Cruciferae (or Brassicaceae) family.
Lines 60-67: plant species are not specified for AtMYB96, QsMYB1, XsMYB44 .
Line 65: Astragalus membranaceus – should be write in italics.
Line 73: to osmosis and MV – no abbreviation decoding.
Material and methods
Line 93: extracted leaf RNA – which method, reagents? The same for cloning procedure (line 94).
Figure 1: some mistakes in latin names (Raphanus sativas for example).
Lines 114-116: The method referred to Zhu [22], according to the map of pGADT7 and pGBKT7 114 vector insert the restriction site EcoRI/BamHI to synthesize primers and amplify the BcMYB44, BcPAP1 and BcEGL3 target genes.
It is not clear what is meant.
The Materials and methods are described insufficiently and poorly correspondent to the Results. For example, the part Subcellular localization.
Lines 102-103: We designed two specific primers, pRI101-BcMYB44-F/R (Table S1), and amplified the target fragment using pEASY-BcMYB44 vector plasmid as a template. No explaining in the text what the vector pEASY-BcMYB44 is, why it was used and what fragment was amplified.
Lines 103-106: The expression vector pRI101 was digested with BamHI/NdeI restriction endonucleases. The amplified fragment was homologous and recombined into pRI101-GFP vector to produce the fusion expression vector, which was transferred into Agrobacterium GV3101. No references for vectors pRI101 and pRI101-GFP, insufficient describing of vector construction and what exactly were inserted in them.
Lines 184-185: The nucleus marker was used to mark the position of the nucleus and emit a clear red fluorescent signal under the RFP channel.
Which nucleus marker was used, how the material for microscopy was prepared – all this was not mention in the Methods. On fig. 2 the scale bars on upper and lower lines of pictures are different (20 and 50 micrometers, despite 50 micrometers in figure legend) making difficult to compare results.
Taking together, it is quite hard to understand how experiment was done and what results were obtained.
The same can be said for other sections.
Only gene and protein abbreviations are given in text.
The part Conclusion is almost identical to the Abstract.
Recommendation: rewriting the manuscript.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
The manuscript describes one of the members of the R2R3 Myb family and its role in inhibiting anthocyanin production and its effect on drought resistance. I like the choice of the experimental subject, Chinese cabbage, because as a representative of the Brassicaceae family it shows variability in leaf coloration and the authors could study contrasting genotypes. On the other hand, this potential remained only partially exploited (VIGS experiments). Furthermore, I would welcome the involvement of transcriptomics and possibly stable Agrobacterium-based transformation, as this should not be a problem for the authors since they have created so many plasmid constructs and the transformation protocol should be available. The authors have nicely described the inhibitory function of BcMyb44 and suggested a contradictory function with flavonol pathway, but there was not done much progress on the regulation of overall pathway branches and the possible role of BcMyb44 as a switch between the branches. Further, the evolutionary or physiological aspect related to the pleiotropic effect could have been discussed more. Nevertheless, the manuscript is worth publishing, it just needs some improvements.
Title: please add the Latin name of the Chinese cabbage to the end of title (to make the abbreviation BcMyb44 clear)
Abstract: specify NJZXI-3' and NJZXI-0' - are they just genotypes, cultivars, new breedings?
Introduction: line 51 - The R2R3Myb family is very large, but is it really clear that repressors dominate? Is this dominance significant? There are many examples of activators. Couldn't you mention, for example, which domains are responsible for the repression? Could you mention some classes of R2R3 Mybs?
Materials and Methods: line 85: specify “five-leaf leaves”. Shouldn't it be “fifth-leaf leaves”?
Chapter 2.3 - tobacco, specify the species: as tobacco is usually considered N. tabacum, but according to your figures it clearly looks like N. benthamiana.
The term "back of tobacco leaf" is very unusual. Substitute with, for example, “the abaxial side of the leaf blade” or similarly.
The term “transient color detection” - wouldn't it be better to use the term “pigment detection” or “transient anthocyanin pathway activation”?
line 148 - please add the specification to gene gun device.
Results:
The total protein formula C1406H2219N421O443S16 is not that interesting. For Figure 1A I missed a clear indication of the different domains - R2, R3 and especially the domain responsible for repression.
Figure 1B – your experimental object is listed there as Brassica rapa and not B. campestris, BcMyb44 could be highlighted.
For section 3.4, the title could mention “protein activity in anthocyanin pathway”, only “color detection” does not sound good.
Fig. 5 - the leaves don’t look like those from N. tabaccum, but N. benthamiana, usually used for agroinfiltration experiments (anyway, the term agroinfiltration could be mentioned also in MM section).
Chapter 3.5 - put VIGS in the title and not just general "gene silencing"
Indeed, the inhibitory function on anthocyanin formation was clearly confirmed in this work. I'm glad that the authors investigated also the genes from non-anthocyanin-specific part of the pathway (PAL, CHS), when studying gene expression, but I think transcriptomics such as RNAseq would provide a more comprehensive picture of what pathways and their branches are affected by the BcMyb44 gene and it would increase the scientific value of the paper.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Thank you for accepting my remarks, I hope it helped to improve your manuscript.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 4 Report
The manuscript was changed according to my comments, but the authors did not always make the changes correctly.
line 52 - "fourth group is mostly" insert the word "represented"
line 88 - treatment was treated? ("provided" would be maybe better)
line 93 - "0.1g leave of fifth leaf material"? delete the word "leave"
lines 134,140 - abaxial side of N. benthamiana - insert the word leaves
Figure 1A - indicate what is R2, what is R3 domain. Compared to other diagrams there is a 2 amino acid shift, but it doesn't matter. If presented myb is a repressor, the authors should show some motif responsible for repression, e.g. EAR motif. i'm sure they will find it (LSLSL)
Figure 1B - Brassica rapa and Brassica campestris are synonyms for chinese cabbage ?
Author Response
Please see the attachment.
Author Response File: Author Response.docx
