Characterization of the HSP70 Gene Family and Its Expression Under Heat Stress in Non-Heading Chinese Cabbage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive genome-wide analysis of the HSP70 gene family in non-heading Chinese cabbage (Brassica rapa ssp. chinensis, NHCC). The study identifies HSP70 genes in the NHCC genome and characterizes their evolutionary relationships, gene structures, conserved motifs, and synteny with related genomes. Both RNA-seq data and qRT-PCR validation indicate that most BrcHSP70 genes are heat-inducible, with the tolerant cultivar showing significantly stronger or sustained induction for a subset of genes.

Major points:

• Figure 1 can add the putative subcellular localization.
• the authors might consider including more distant outgroup sequences (e.g. HSP70s from another Brassicaceae or monocot species) to further validate subgroup delineations, though this is not essential in their phylogenetic approach.
• the authors correctly conclude that whole-genome duplication events have driven HSP70 expansion. To strengthen this section, it would be valuable to perform a duplication age or selection analysis (e.g. Ka/Ks for each duplicated pair). Calculating Ka/Ks here would reveal if these NHCC duplicates are under strong purifying selection (implying conserved function) or diversifying (potential neo- or sub-functionalization). 
• Consistency in naming (sometimes “BrHSP70” vs “BrcHSP70” appears) should be checked. 
• the cross-species synteny figure (Fig 6), while valuable, may not be essential for the main message. It largely confirms known relationships (Brassica vs Arabidopsis divergence). The authors might consider moving Figure 6 to Supplementary Materials to streamline the main text, unless they plan to discuss specific ortholog pairs of interest.
• expression data (rnaseq, qPCR): It would strengthen the argument if the authors provide statistical analysis of the expression differences – for example, log₂ fold-change and significance (P-value or FDR) for each gene between the two cultivars. Were the RNA-seq replicates analyzed for differential expression? Right now, terms like “upregulated” vs “downregulated” are used, but it’s not explicit if these passed a statistical threshold or are descriptive. Including a supplemental table of fold-changes and q-values for each gene at 6 h and 24 h (tolerant vs sensitive) would back up the claims. Additionally, a brief comment on basal expression levels (time 0) would be useful – did ‘SHI’ and ‘Aijiaohuang’ start with similar HSP70 transcript levels, or does the tolerant line inherently express some HSP70s more even before stress? For example, if certain genes are basally higher in ‘SHI’, that could contribute to its thermotolerance. If such data are available from time 0 replicates, the authors should mention any notable constitutive differences. In terms of the heat treatment itself, 38 °C for up to 24 h is a reasonably severe stress for Brassica. The paper would benefit from reporting any phenotypic effects observed (leaf wilting, recovery, etc., if noted) to tie gene expression to plant performance. Even if qualitative, noting that ‘SHI’ plants tolerated the 24 h heat better than ‘Aijiaohuang’ (if true) would create a more tangible link between gene expression and phenotype.
• The promoter analysis reveals an abundance of hormone and stress-responsive cis-regulatory elements in HSP70 promoters, which is an important addition to understanding regulatory mechanisms. Critically, one key cis-element is conspicuously not discussed: the Heat Shock Element (HSE). HSEs are the canonical binding sites for Heat Shock Transcription Factors (HSFs) and are known to drive HSP gene induction under heat stress. It is surprising that HSE motifs were not explicitly mentioned, since virtually all heat-inducible HSP70 genes contain HSEs in their promoters. Please add this analysis.
• The authors should reference comprehensive reviews and articles about heat perception and signaling such as 10.1016/j.tplants.2022.03.006, 10.1016/j.tibs.2022.05.004, and more recent publication about the role of HSP70 in agriculture/global warming.

Minor points:

• Figure Legends: Expand some legends for self-containment. For instance, Fig 1’s legend should specify what the colored circles at nodes mean (the text mentions bootstrap and posterior probability support, but the legend snippet suggests they are indicated by colored circles – ensure it’s clear which color = which support range). Fig 5’s legend should clarify that colored lines inside the circle connect duplicated gene pairs (assuming that’s the case). Fig 8 and Fig 9 legends should list the full names of the cis-elements abbreviated.
• The statement “No new data were created or analyzed in this study” is puzzling, since the authors did perform new qRT-PCR experiments and new analyses of a published RNA-seq dataset. This statement might be a template error. It should be corrected to indicate that all data supporting the findings are provided (sequence data from public genome resources, RNA-seq data from PRJNA1030162, and qPCR results in this article or available on request). This is important for transparency.
• In the Methods, include details about biological replicates and statistical tests. For RNA-seq, how many replicates per condition were in the dataset? For qRT-PCR, how many biological and technical replicates were used, and what reference gene and analysis method (e.g., ΔΔCq method) was applied?  In the Results, when saying “significantly differed” or “upregulated/downregulated”, if this is based on a statistical threshold (e.g., >2-fold and p<0.05), mention that criterion.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I have reviewed your manuscript "Characterization of the Hsp70 Gene Family and Its Expression under Heat Stress in Non-Heading Chinese Cabbage", submitted for publication in Horticulturae.

After reading your manuscript I can tell that your research is well conducted, the research design and scientific reasoning are simple but canonical and robust, and the results are decently presented and well discussed. The manuscript displays minor flaws mostly in terms of grammar, sentence structure, use of italic letters and similar. For the revision of your manuscript please follow my comments as given below:

• Hsp70 genes and Hsp70 proteins: Throughout your manuscript, you randomly use the italic letters and plain text letters, as well as designations "genes" or "proteins" when referring to Hsp70 genes or Hsp70 proteins. Examples include: "Hsp70 genes" (line 99, you are indeed referring to genes, but using plain text letters), "Hsp70 proteins" (line 162 and line 331, you are indeed referring to proteins, but using italic letters), "Hsp70 proteins" (line 169 and line 177, here you are referring to the Hsp70 genes because you are talking about the phylogenetic tree. You are also correctly using the italic letters because you are actually talking about genes, but instead of "Hsp70 genes" you wrote "Hsp70 proteins"). Please thoroughly revise the entire text of your manuscript (including Tables and Figure captions) to make sure that you always correctly refer to genes or to proteins, including the correct use of italic letters or plain text, depending on context. Another remark is that you use first-capital letter (Hsp70 or Hsp70) or all-caps (HSP70 or HSP70) interchangeably. Since first-capital (Hsp70 or Hsp70) is in more widespread use, I suggest to replace all-caps (HSP70 or HSP70) with first-capital (Hsp70 or Hsp70) for both genes and proteins throughout the manuscript, including Tables and Figure captions.

 

• other remarks:
• Abstract:
  • line 16: Please replace "remain unclear" with "have not been investigated". For a phenomenon to "remain unclear", it is necessary that the phenomenon has already been investigated but unsuccessfully, whereas, as much as I understand, your endeavor at investigating the Hsp70 family in bok choy is the first such research project, even though a similar project has been carried out in HCC by Tabusam and co-authors.
  • line 19: "pekinensis" should be written in lowercase letters. A subspecies name, just like species name, is always written in lowercase; only the genus name is written with a capital letter.
  • line 22: "cis" in "cis-acting" should be written in italic.

 

• Introduction:
  • line 47: Please replace "dryness" with "drought"
  • line 53: Please replace "duties" with "functions". "Duty" requires conscious responsibility, whereas proteins only have functions.
  • line 58: Please delete "known scientifically as". This is newspaper language, not language for a scientific paper. You should write "Non-heading Chinese cabbage (NHCC, Brassica rapa chinensis)...". Also please note that "subsp." should be written in plain text.
  • line 70-71: Please replace with: "However, the characteristics of these genes in NHCC have not yet been explored." (See previous comment for line 16.)
• Research aim and objectives:
  • line 72-73: The aim of your research is not well defined. "To perform a comprehensive examination of the Hsp70 gene family in NHCC" is a task, not a project aim, because it is activity-oriented and not outcome-oriented. The aim could be defined as "to clarify the structural and functional properties of the members of the Hsp70 gene family in NHCC".
  • line 73: What you further list as (1), (2) and (3) are tasks, not objectives. I suggest to simply replace the word "objectives" with "tasks" at line 73.

 

• Materials & Methods:
  • line 132: A citation of your previous research under which this dataset was generated, would be obligatory since the readers will also want to get to know the other findings of that research, which is tightly related to the present one. If your previous paper is not on the current References list, add it, and please make sure to thoroughly re-number all the subsequent references in the manuscript, so as to prevent a confusion among the cited papers to arise from the insertion of this reference.
  • line 145-148: Please state the model and manufacturer of the thermocycler machine in which the qPCR analysis was performed. Also please state which housekeeping gene was used as a reference gene for the 2^-ΔΔCt

 

• Results:
  • line 160: "chinensis" should be written in lowercase (same comment as earlier, for line 19).
  • Table 1, header: the letter p in "pI" for the isoelectric point should be written in lowercase.
  • line 169: Please add the word "of": "Phylogenetic analysis of Hsp70 genes". (Also, you are talking about the genes here, not proteins.)
  • line 177: Same.
  • line 194-195: "Notably, motif 9..." - this sentence is nonsensical. Looking at Figure 2B, I imagine that you wanted to say that motif 9 is present in all the BrcHsp70 proteins except in one of the Group 5 members. (I suppose you meant BrcHsp70-18.3.)
  • line 199: Please delete "Analysis of the" from the Figure caption.
  • line 199: Also please replace "evolutionary tree" with "phylogenetic tree".
  • line 219: There are multiple mistakes in the text at this line. Firstly, at chromosome A01 there are five (and not seven) Hsp70 Secondly, the sentence ends with "and on Chr1, Chr3, and Chr6" but chromosomes 1 and 3 are already mentioned in the sentence since these refer to A01 and A03. Please double-check the Figure and thoroughly revise the entire sentence.
  • line 256: I suggest to replace "potential functional conservation" with "likely functional conservation", which would be a stronger statement. I feel like functional conservation in this case is very, very likely, so there is no need for an over-cautious statement.
  • line 263: Please add the word "only": "encompassing not ONLY..."
  • line 267-273: Figure 8 needs to be cited already in this paragraph because reading it at first, the reader gets an immediate impression that the results described in this paragraph are not shown in the manuscript. The subsequent citation at line 278 comes too late.
  • line 274-277: Same comment regarding a needed citation of Figure 9.
  • Figure 7, caption: Please expand the Figure caption to explain the meaning of the Figure more thoroughly, including the meaning of the use of green, brown, and red tones for the boxes.
  • line 293: A reference to your previous work, under which the dataset was generated, should be repeated here.
  • line 302-303: "In general, the expression of 'SHI'..." - Looking at Figure 10, I actually had the opposite impression. It looks like most genes are more readily upregulated in 'Aijiaohuang' (at 6 h), but they are also downregulated at 24 h, whereas in the thermotolerant genotype the upregulation of most genes seems to occur more slowly, but is more persistant, so that the genes are still upregulated at 24 h. This is important, since heat stress normally occurs in the form of long-lasting heat waves, so the ability of the plant to maintain a long-term upregulation of heat tolerance genes is crucial for its adaptation to heat.
  • Figure 10, caption: The Figure caption needs to contain the information that the data represented in the heatmap come from the RNA-Seq analysis. Also, the numbered reference of the published paper under which the dataset was generated, should be provided here within the Figure caption.
  • Figure 11, caption: Please replace "and stressful times" with "and at different duration of exposure to heat stress".

 

• Discussion:
  • line 327: Please delete "renowned as" and leave just "HSPs are versatile molecular chaperones". HSPs (plain text) are not "renowned". A person or a city may be renowned, but not a protein.
  • line 335: Vigna, as a genus name, needs to be written with a capital V.
  • line 339-340: The previous study by Tabusam et al. (which you are citing at this point) revealed 28 members of the Hsp70 gene family in HCC, but the in silico analysis that you performed in the present research identified 30 of them, as mentioned that at line 172. Obviously, your analysis was more permissive and identified two more Hsp70 genes in HCC in addition to the 28 genes identified by Tabusam and co-authors. This point is worth making a short remark; otherwise, the readers may become confused with apparently contradictory information that you are giving in your Introduction and Discussion, as compared to what is said in the Results section.
  • line 356: Please insert an empty space in "ofmotifs"
  • line 362: Please replace "on the identical chromosome" with "on the same chromosome". The words "same" and "identical" are not synonyms. For example, two twin brothers may be identical (impossible to spot a difference between) but they are two separate entities, they are not the same person. Here, you are talking about two genes that are located literally on the same chromosome.
  • line 374: "cis" in "cis-acting" should be written in italic
  • line 385-386: Please replace "of plant adaptive responses to drought" with "of the adaptive responses of NHCC to drought". These mechanisms are already very much elucidated in plants, but not in NHCC, so this is where your novelty lies. Also, it might be worth noting that drought and heat stress often co-occur in nature, which explains the versatility of the BrcHsp70 promoters in the responses to these two types of stress.
  • line 391: please add "they were" ("whereas they were downregulated..."). Also, it is worth noting here that the upregulation of these genes in the 'Aijiaohuang' variety lasts shorter.
  • line 406: Please delete the word "differential". "Different differential" equals just "different".

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,
Thank you for the substantial improvements made in the revised manuscript. The updated Figure 6 clearly shows the distribution of canonical heat‑shock elements (HSEs); however, these new data are not yet reflected in the text. To complete the revision, please insert 2–3 concise sentences (immediately after the promoter‑motif paragraph) that (i) state how many BrcHSP70 promoters harbour HSEs, (ii) give the typical copy‑number range, and (iii) note any subgroup enrichment. And in the Discussion, please add 2–3 sentences explaining the functional relevance—e.g., links to HSF binding, the rapid induction of specific BrcHSP70 genes, and how HSE abundance might underpin the expression differences observed between the heat‑tolerant and heat‑sensitive cultivars.

Moreover there are still some typo and proofreading to do, but this is very minor. 
Best regards,

Author Response

"Please see the attachmen

Author Response File: Author Response.pdf