Genome-Wide Identification and Analysis of the WRKY Gene Family in the Xerophytic Evergreen Ammopiptanthus nanus
Round 1
Reviewer 1 Report
The manuscript has been improved and comments were incorporated. Please in the figure open up SE as standard error (at least when first time it appear).
However, I wonder (I have concerns) that why AnWRKY19, AnWRKY 56 (from previous version, Figure 5) and AnWRKY6, AnWRKY12 (from previous version, Figure 6) are not presented anymore? while they are present in current Table 1 and they have been included in the previous version .
The quality of the figures can be much improved.
Good luck!
Author Response
Response to Reviewer 1 Comments
Point 1: Please in the figure open up SE as standard error (at least when first time it appear).
Response 1: We have changed “SE” to “standard error (SE)” when first time it appeared in line 278.
Point 2: However, I wonder (I have concerns) that why AnWRKY19, AnWRKY56 (from previous version, Figure 5) and AnWRKY6, AnWRKY12 (from previous version, Figure 6) are not presented anymore? while they are present in current Table 1 and they have been included in the previous version .
Response 2: Based on the suggestions of previous reviewer, we have renamed 63 AnWRKY genes using another naming method. AnWRKY19, AnWRKY56 (from previous version, Figure 5) and AnWRKY6, AnWRKY12 (from previous version, Figure 6) have been renamed to AnWRKY9, AnWRKY62 (in current version, Figure 4) and AnWRKY26, AnWRKY34 (in current version, Figure 5), respectively. At the same time, we have made the corresponding modification to the gene names in the text.
Reviewer 2 Report
General comments: I enjoyed reading a paper of a non-model organism that show what can be done by having the genome assembled. I find the results of this paper are a good base for further research for breeding (if possible) A. nanus. As someone not familiar with this part of the tree of life, it would be nice to also include a phylogenetic tree that include some model organisms, some crops and some trees to have a better context that can help to interpret the conservation of the gene families. The language still requires some polishing, but it is possible to understand the meaning of the authors. Mayor.- The document was sent with notes between the authors. Texts to review must never include those notes.
- The abstract claims to make a new classification of the WRKY families, but the introduction mentions the existing classification (based on a reference to Vitis vinifera). It should be clear that this work is following (or not) the previously stablished classification.
- Section 2.1, show how many. Proteins you filter out on each step. For example, how many sequences you have before and after the low-reliability and redundant sequences were filtered?
- Methods, in general. Show the scripts and the exact commands used, to improve the reproducibility of your study.
- Line 194-198: What may be the case for this difference in the distribution of WRKY gene families? Does this has a relationship to when this family separated from better know organisms? Or is it because there are still missing genes in the assembly? Can you find genes that are not predicted using the conserved primers (based on the length of the bands?)
- I like the validation of the wrky48, and I appreciate that working with An is not as standard as working with At, but I wonder if you can produce in the discussion a roadmap of testing this (and other genes) on your target organism.
- Include an introductory phylogenetic tree to give the context of the organism. It doesn’t have to be from the WRKY genes, but something more general (as mentioned above)
- Line 35: Should read “are used in traditiona Chinese medecine”. Maybe out of the scope, but it could be speculated the role of some genes in the compounds that produce the medicinal effect.
- Line 50, check grammar.
- Line 93. Instead of “cherished”, the phrase “It is regarded as a plan that can withstand… “ is more native
Author Response
Response to Reviewer 2 Comments
Point 1: The document was sent with notes between the authors. Texts to review must never include those notes.
Response 1: In the previous modification, the reviewers could not see the figures I sent, and another author inserted the images to solve this problem, that may cause the appearance of these notes. The notes are formal modifications to the text for reviewers instead of other authors. In the current version, these notes have been deleted.
Point 2: The abstract claims to make a new classification of the WRKY families, but the introduction mentions the existing classification (based on a reference to Vitis vinifera). It should be clear that this work is following (or not) the previously stablished classification.
Response 2: The classification of the WRKY families here is following the previously stablished classification (not based on a reference to Vitis vinifera), which has clearly explains in line 58-63 of the text, and AnWRKY has the same classification method. As for VviWRKY40, it was just a introduction of research progress on a new function of a WRKY gene, which was also suggested by a previous reviewer.
Point 3: Section 2.1, show how many. Proteins you filter out on each step. For example, how many sequences you have before and after the low-reliability and redundant sequences were filtered?
Response 3: We only filtered out proteins after conserved domain analysis with SMART software. Except for proteins with extremely low credibility, only 2-3 proteins without the typical WRKY domain have been screened out. After filtering, we finally obtained 63 AnWRKY proteins.
Point 4: Methods, in general. Show the scripts and the exact commands used, to improve the reproducibility of your study.
Response 4: In this study, except for the software mentioned in the article, no scripts and the exact commands were used. And all the results of this research can be reproduced via these ordinary software.
Point 5: Line 194-198: What may be the case for this difference in the distribution of WRKY gene families? Does this has a relationship to when this family separated from better know organisms? Or is it because there are still missing genes in the assembly? Can you find genes that are not predicted using the conserved primers (based on the length of the bands?)
Response 5: The genome of A. nanus has been sequenced with a good completeness. In theory, after searching in the whole genome, the genes we obtained are all AnWRKY genes. And it can be seen from Table 2 that the proportion of AnWRKY in each subfamily is basically consistent with other WRKYs. So this difference in distribution should stem from the classification or evolution of the WRKY family. Initially, the WRKY family was divided into three groups (I, II and III) based on the phylogenetic analysis and the features of the WRKY domain. Group I proteins usually contain two WRKY domains and C2H2 type zinc finger motifs in the N- and C-terminal. Some group II WRKY genes are likely to be evolved from group I WRKY genes by losing either the N- or C-terminal WRKY domain [1]. The replacement of the conserved His residue with a Cys residue (C2H2 to C2HC) in the zinc finger motif might result in the evolution of group III WRKY genes [2]. Subsequently, group II splits up into five distinct subgroups, according to domain feature, the phylogenic tree, and the intron pattern [1]. Therefore, a more detailed classification may separate the members of the WRKY family.
Point 6: I like the validation of the wrky48, and I appreciate that working with An is not as standard as working with At, but I wonder if you can produce in the discussion a roadmap of testing this (and other genes) on your target organism.
Response 6: We added the sentences in line 396-399 of discussion as followings: In order to further verify this function, the exogenous AnWRKY48 may be overexpressed in different model plants for testing the insect resistance ability. In the other hand, establishing a tissue culture system of A. nanus and transient expression technology could be used to analyze the gene functions.
Point 7: Include an introductory phylogenetic tree to give the context of the organism. It doesn’t have to be from the WRKY genes, but something more general (as mentioned above).
Response 7: There were few studies on A. nanus before. Recently, researchers have conducted a systematic study on the Chitinases [3] and AP2/ERF [4] gene families of A. nanus. Compared to model organisms, genes in A. nanus have similar distributions and higher conservation, and we got a unanimous results in AnWRKY (Figure S1 in the article).
Point 8: Line 35: Should read “are used in traditional Chinese medicine”. Maybe out of the scope, but it could be speculated the role of some genes in the compounds that produce the medicinal effect.
Response 8: We have changed the original sentence to “In addition, it can be used as oil and some genes in the branches and leaves may produce the medicinal effect” in line 35.
Point 9: Line 50, check grammar.
Response 9: We have changed the original sentence to “The WRKY genes are transcriptional regulators that play important roles in plant defense and stress responses” in line 51-52.
Point 10: Line 93. Instead of “cherished”, the phrase “It is regarded as a plant that can withstand…” is more native
Response 10: According to the suggestion, we have changed it to “It is regarded as a plant that can withstand…” in line 93-94.
References
- Eulgem, T.; Rushton, P.; Robatzek, S.; Somssich, I. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000, 5, 199-206.
- Xie, Z.; Zhang, Z.; Zou, X.; Huang, J.; Ruas, P.; Thompson, D.; Shen, Q.J. Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol. 2005, 137, 176-189.
- Cao, S.; Wang, Y.; Li, Z.; Shi, W.; Gao, F.; Zhou, Y.; Zhang, G.; Feng, J. Genome-Wide identification and expression analyses of the chitinases under cold and osmotic stress in Ammopiptanthus nanu. genes 2019, 10, 472.
- Cao, S.; Wang, Y.; Li, X.; Gao, F.; Feng, J.; Zhou, Y. Characterization of the AP2/ERF transcription factor family and expression profiling of DREB subfamily under cold and osmotic stresses in Ammopiptanthus nanus. Plants 2020, 9, 455.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
All of my major comments were adequately addressed. I do not have any additional comments for the authors.
Author Response
Dear reviewer,
Thank you very much for your approval!
Reviewer 2 Report
1. Line 34-36: Check English. “This species has completed genome sequencing in
2018, the final size of the assembled A. nanus genome was close to the 889 Mb,
and the gene annotation was identified as complete by BUSCO [1].”
2. Line 55-57: rephrase it please. “The first reported WRKY gene was identified in
sweet potato (Ipomoea batatas) in 1994 and regulates the expression of storage
protein and β-amylase-related genes [8]”.
3. Line 88: sentence cannot start with And! Please correct this. “…And ABA
could…”.
4. Line 89: Why you wrote this sentence. Please rephrase it. “This will widen our
knowledge of WRKY in grape breeding [23].”
5. Line 95-98: Nice sentence!
6. Figure 2 can be presented as supplementary material in a clear manner as here
it is difficult get information.
7. Fig 5 and 6:
In Figure 5: Is really the expression of AnWRKY19 in roots between HW and
control significantly different? Please re-check!
The expression of AnWRKY56 in roots between HW and control seems
significantly different but there is no sign. Please pay more attention.
In Figure 6, the expression of AnWRKY6, AnWRKY12, AnWRKY35,
AnWRKY53 berween leaves in drought conditions and control samples are
significantly different but it is not marked!
8. Lines 479-480: I suggest you open/explain calmodulin as “calcium-modulated
protein”.
9. Lines 487-488: Still I find this sentence confusing. Please modify it.
“Similar phenomena were happened in Arabidopsis and cucumber, and these
genes included AtWRKY50, 51, 59, and CsWRKY41, 44, 54, which also
belonged to subgroup IIc.”
10. Lines 537-539 and 540-542 is REPEATED!
11. Line 538: Should you use other verb than establish? “…In this study, we
established that…”.
12. Lines 542-544: Please rephrase the sentence. Verb is missing!
“In other species [39,41,42], the studies on WRKY transcription factors mainly
through the abiotic or pathogenic treatment, but rarely for herbivory.”
13. Lines: 544-545: Unclear sentence. “However, the insect resistance mechanism
of individual WRKY genes has been deeply studied”.
14. Line 548: I would add “In the current study” before “nine genes with obvious
changes”.
15. Lines 557-558: Please rephrase it or write in other words. “Among these genes,
there are different trends about up or downregulated (Figure 6), indicating variety
roles in this process”.
16. Line 562: Compared to instead of “compared with”.
17. Line 573: Unclear sentence. What do you want to say by that “even those with
low expression levels in some tissues?”
Good Luck!
Author Response
Response to Reviewer 2 Comments
Please note that the "line number" in this file corresponds to the Word version, not the PDF version.
Point 1: Line 34-36: Check English. “This species has completed genome sequencing in 2018, the final size of the assembled A. nanus genome was close to the 889 Mb, and the gene annotation was identified as complete by BUSCO [1].”
Response 1: “This species has completed genome sequencing in 2018, the final size of the assembled A. nanus genome was close to the 889 Mb, and the gene annotation was identified as complete by BUSCO [1]” has been changed into “The genome of A. nanus was sequenced in 2018 and the final size of the assembled this genome was close to the 889 Mb, in which the gene annotation integrality was estimated by BUSCO [1]” in line 40-42.
Point 2: Line 55-57: rephrase it please. “The first reported WRKY gene was identified in sweet potato (Ipomoea batatas) in 1994 and regulates the expression of storage protein and β-amylase-related genes [8]”.
Response 2: “The first reported WRKY gene was identified in sweet potato (Ipomoea batatas) in 1994 and regulates the expression of storage protein and β-amylase-related genes [8]” has been changed into “The first reported WRKY gene was identified in sweet potato (Ipomoea batatas) in 1994, which was reported to be regulated the expressions of sporamin and β-amylase-related genes [9]” in line 59-61.
Point 3: Line 88: sentence cannot start with And! Please correct this. “…And ABA could…”.
Response 3: The previous sentence has been changed to “In addition, recent studies showed that VviWRKY40 expression pattern was negatively correlated with that of VviGT14 in grape berries (Vitis vinifera L.) and reported that ABA could downregulate the transcript level of VviWRKY40 in order to control monoterpenoid glycosylation [24]” in line 90-93.
Point 4: Line 89: Why you wrote this sentence. Please rephrase it. “This will widen our knowledge of WRKY in grape breeding [23].”
Response 4: The previous sentence has been deleted.
Point 5: Line 95-98: Nice sentence!
Response 5: Thanks a million!
Point 6: Figure 2 can be presented as supplementary material in a clear manner as here it is difficult get information.
Response 6: Figure 2 has been placed in supplemental figure S1.
Point 7: Fig 5 and 6:
In Figure 5: Is really the expression of AnWRKY19 in roots between HW and control significantly different? Please re-check! The expression of AnWRKY56 in roots between HW and control seems significantly different but there is no sign. Please pay more attention.
In Figure 6, the expression of AnWRKY6, AnWRKY12, AnWRKY35, AnWRKY53 between leaves in drought conditions and control samples are significantly different but it is not marked!
Response 7: I agree with you, because I had the same idea. We re-checked the data, the following is the original data related to this problem. After we test the homogeneity of variance, the result of t-test is scientific and credible. This may be due to large errors between biological replicates.
|
AnWRKY19 |
Sample |
Relative Expression |
p-values |
Statistical Significance
|
|
Biological Replicate 1 |
control-roots |
1.019286754 |
0.017596830 |
* |
|
HW-roots |
1.167464699 |
|||
|
Biological Replicate 2 |
control-roots |
1.022930601 |
||
|
HW-roots |
1.172964773 |
|||
|
Biological Replicate 3 |
control-roots |
1.006421572 |
||
|
HW-roots |
1.248582944 |
|||
|
|
|
|
|
|
|
AnWRKY56 |
|
|
|
|
|
Biological Replicate 1 |
control-roots |
0.832522862 |
0.009153068 |
** |
|
HW-roots |
0.199390235 |
|||
|
Biological Replicate 2 |
control-roots |
1.000342777 |
||
|
HW-roots |
0.227217155 |
|||
|
Biological Replicate 3 |
control-roots |
0.567013085 |
||
|
HW-roots |
0.171563314 |
|||
|
|
|
|
|
|
|
AnWRKY6 |
|
|
|
|
|
Biological Replicate 1 |
control-leaves |
1.225946086 |
0.050019930 |
N/A |
|
drought-leaves |
2.123254068 |
|||
|
Biological Replicate 2 |
control-leaves |
1.007955124 |
||
|
drought-leaves |
1.654114472 |
|||
|
Biological Replicate 3 |
control-leaves |
1.000366878 |
||
|
drought-leaves |
3.127068826 |
|||
|
|
|
|
|
|
|
AnWRKY12 |
|
|
|
|
|
Biological Replicate 1 |
control-leaves |
1.013858021 |
0.179702231 |
N/A |
|
drought-leaves |
0.410141933 |
|||
|
Biological Replicate 2 |
control-leaves |
2.480881460 |
||
|
drought-leaves |
0.392828406 |
|||
|
Biological Replicate 3 |
control-leaves |
0.426455227 |
||
|
drought-leaves |
0.102418803 |
|||
|
|
|
|
|
|
|
AnWRKY35 |
|
|
|
|
|
Biological Replicate 1 |
control-leaves |
1.029139918 |
0.115620193 |
N/A |
|
drought-leaves |
7.440357577 |
|||
|
Biological Replicate 2 |
control-leaves |
0.612199867 |
||
|
drought-leaves |
2.422681640 |
|||
|
Biological Replicate 3 |
control-leaves |
2.541028974 |
||
|
drought-leaves |
12.976846113 |
|||
|
|
|
|
|
|
|
AnWRKY53 |
|
|
|
|
|
Biological Replicate 1 |
control-leaves |
0.282342793 |
0.325368562 |
N/A |
|
drought-leaves |
0.171288613 |
|||
|
Biological Replicate 2 |
control-leaves |
1.001819112 |
||
|
drought-leaves |
0.283391084 |
|||
|
Biological Replicate 3 |
control-leaves |
0.218696913 |
||
|
drought-leaves |
0.076929394 |
Point 8: Lines 479-480: I suggest you open/explain calmodulin as “calcium-modulated protein”.
Response 8: According to the suggestion, “calmodulin” has been changed into “calcium-modulated protein” in line 494.
Point 9: Lines 487-488: Still I find this sentence confusing. Please modify it. “Similar phenomena were happened in Arabidopsis and cucumber, and these genes included AtWRKY50, 51, 59, and CsWRKY41, 44, 54, which also belonged to subgroup IIc.”
Response 9: “Similar phenomena were happened in Arabidopsis and cucumber, and these genes included AtWRKY50, 51, 59, and CsWRKY41, 44, 54, which also belonged to subgroup IIc.” has been changed into “Similar phenomena were reported in Arabidopsis and cucumber [7,25]” in line 501-502.
Point 10: Lines 537-539 and 540-542 is REPEATED!
Response 10: Repeated sections have been deleted.
Point 11: Line 538: Should you use other verb than establish? “…In this study, we established that…”.
Response 11: “Established” has been changed into “demonstrated” in line 502.
Point 12: Lines 542-544: Please rephrase the sentence. Verb is missing! “In other species [39,41,42], the studies on WRKY transcription factors mainly through the abiotic or pathogenic treatment, but rarely for herbivory.”
Response 12: “In other species [39,41,42], the studies on WRKY transcription factors mainly through the abiotic or pathogenic treatment, but rarely for herbivory.” has been changed into “In other species [39,41,42], the studies on WRKY transcription factors were mainly focused on abiotic or pathogenic treatment, but few studies on herbivory.” in line 546-547.
Point 13: Lines: 544-545: Unclear sentence. “However, the insect resistance mechanism of individual WRKY genes has been deeply studied”.
Response 13: “However, the insect resistance mechanism of individual WRKY genes has been deeply studied” has been changed into “Only individual WRKY genes have been reported to be related to insect resistance” in line 547-548.
Point 14: Line 548: I would add “In the current study” before “nine genes with obvious changes”.
Response 14: “In the current study” has been added before “nine genes with obvious changes” in line 552.
Point 15: Lines 557-558: Please rephrase it or write in other words. “Among these genes, there are different trends about up or downregulated (Figure 6), indicating variety roles in this process”.
Response 15: “Among these genes, there are different trends about up or downregulated (Figure 5), indicating variety roles in this process” has been changed into “These upregulated or downregulated genes might be positively or negatively correlated with drought resistance, respectively.” in line 561-562.
Point 16: Line 562: Compared to instead of “compared with”.
Response 16: According to the suggestion, “compared with” has been changed into “compared to” in line 566.
Point 17: Line 573: Unclear sentence. What do you want to say by that “even those with low expression levels in some tissues?”
Response 17: The previous sentence has been changed to “Indeed, the AnWRKY genes could make corresponding response according to different stresses.” in line 576-577.
Reviewer 3 Report
The introduction section lacks background information about of the research plant, describe its agronomic values and findings should be related to agronomic practices to be considered for this journal. Recent and prominent literature on the plant WRKY research are missing. Nomenclature of teh WRKYs should follow Arabidopsis.
Line 145: what doe you mean by low quality. Clarify in writing
Line 150: did you used the motif sequence or whole protein sequences for the phylogenetic analysis.
Line 230-231: “The AnWRKY genes were 231 named AnWRKY1 to AnWRKY63.” It is not clear how they were named. Elaborate on the nomenclatural determination. If it is not done based on the homology assessment relative to AtWRKYs, current nomenclature becomes useless.
Line 260: Figure 1-- Is the analysis based on the WRKY domains or the full-length Amino Acid sequences? Follow how Eugen et al, and then Ruston et al (2005, 2010, 2015) for the nomenclature.
Author Response
Response to Reviewer 3 Comments
Please note that the "line number" in this file corresponds to the Word version, not the PDF version.
Point 1: The introduction section lacks background information about of the research plant, describe its agronomic values and findings should be related to agronomic practices to be considered for this journal. Recent and prominent literature on the plant WRKY research are missing. Nomenclature of teh WRKYs should follow Arabidopsis.
Response 1: We have added some introductions to the background information of A. nanus in the first paragraph. Just like: “It could be a good sand fixing plant with salt alkali, drought and sand resistances. Moreover, with beautiful tree shape, many butterfly shaped tubular flowers, golden yellow corolla and white fluff on the twigs, it could be an ideal greening tree species. In addition, it can be used as oil and its branches and leaves can be used for traditional Chinese medicine. It has the effect of dispelling wind, warming and dampness, promoting blood circulation and dispersing blood stasis from the perspective of traditional Chinese medical science. In concision, it has ecological, agronomic, and medicinal value”.
Recent references are as follows:
|
Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub. 2018, Research progress on rare and endangered plant Ammopiptanthus nanus. 2019, The WRKY transcription factor family in model plants and crops. 2017 Overexpression of a Malus baccata WRKY transcription factor gene (MbWRKY5) increases drought and salt tolerance in transgenic tobacco. 2018. Rice WRKY11 plays a role in pathogen defense and drought tolerance. 2018 VviWRKY40, a WRKY transcription factor, regulates glycosylated monoterpenoid production by VviGT14 in grape berry. 2020 Genome-wide identification and characterization of cacao WRKY transcription factors and analysis of their expression in response to witches' broom disease. 2017 Injury activates Ca2+/calmodulin-dependent phosphorylation of JAV1-JAZ8-WRKY51 complex for jasmonate biosynthesis. 2018 AtWRKY40 functions in drought stress response in Arabidopsis thaliana. 2018, |
Prominent references are as follows:
|
The WRKY superfamily of plant transcription factors. Trends Plant Sci. The WRKY transcription factor family in model plants and crops. Crit. Rev. Plant Sci. Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. Embo J. Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell Induced plant defenses in the natural environment: Nicotiana attenuata WRKY3 and WRKY6 coordinate responses to herbivory. Plant Cell A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. Plant Cell Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. Injury activates Ca2+/calmodulin-dependent phosphorylation of JAV1-JAZ8-WRKY51 complex for jasmonate biosynthesis. Mol. Cell |
Indeed, A. nanus has no prominent literature to cite recently. In addition, the literature on software and identification in other species has to be cited.
For nomenclature issues, please see Point 4.
Point 2: Line 145: what doe you mean by low quality. Clarify in writing.
Response 2: Low quality means that the reliability is low, that is, the E value is far greater than 0.001. “Low-quality” has been changed into “low-reliability” in line 199. Since this concept is common, I didn’t give too much explanation in the article.
Point 3: Line 150: did you used the motif sequence or whole protein sequences for the phylogenetic analysis
Response 3: To examine the phylogenetic relationships among AnWRKY proteins, we constructed a phylogenetic tree based on multiple sequence alignment of 63 AnWRKY and 20 AtWRKY proteins (as references) using MEGA 7.0 software. This “protein” refers to the whole protein sequences. It's just that the protein of group I was divided into two parts: N-terminal and C-terminal. Therefore, the previous sentence has been changed into “A phylogenetic tree was constructed with the whole WRKY proteins of Arabidopsis and A. nanus using the neighbor-joining method in MEGA 7.0 software” in line 127.
Point 4: Line 230-231: “The AnWRKY genes were 231 named AnWRKY1 to AnWRKY63.” It is not clear how they were named. Elaborate on the nomenclatural determination. If it is not done based on the homology assessment relative to AtWRKYs, current nomenclature becomes useless.
Response 4: On this issue, we thought carefully at that time. First of all, we constructed a phylogenetic tree for all AnWRKY and AtWRKY proteins and planned to name them according to their genetic relationship. However, we found four situations, 1) several AnWRKY genes clustered with one AtWRKY gene; 2) one AnWRKY gene with some AtWRKY genes clustered together; 3) some AnWRKY genes clustered without AtWRKY gene; 4) no AnWRKY gene clustered with AtWRKY genes. So, it's going to be hard to name it according to the traditional rules. Also, we found that it's not based on genetic relationship with AtWRKY in cucumber (Cucumis sativus) [1], grape (Vitis vinifera) [2], wheat (Triticum aestivum L.) [3], apple (Malus domestica Borkh.) [4], physical nut (Jatropha curcas L.) [5], Chinese cabbage (Brassica rapa ssp. Pekinensis) [6], rubber tree (Hevea brasiliensis) [7], peanut (Achis hypogaea L.) [8], shrib willow (Salix suchowensis) [9] and so on. In addition, most of the above species are named according to the order on the chromosomes. Unfortunately, the chromosome information data of A. nanus has not been released. We got in touch with the team that sequenced the genome of A. nanus, but they were working on another paper and couldn't give me the data yet. So, “The AnWRKY genes were named AnWRKY1 to AnWRKY63.” has been changed into “According to the classification of subfamily, AnWRKY genes were named AnWRKY1 to AnWRKY63.” in line 186-187.
Point 5: Line 260: Figure 1-- Is the analysis based on the WRKY domains or the full-length Amino Acid sequences? Follow how Eugen et al, and then Ruston et al (2005, 2010, 2015) for the nomenclature.
Response 5: The analysis in Figure 1 is based on the full-length of the Amino Acid sequence. According to research requirements, the protein of group I was divided into two parts: N-terminal and C-terminal. For nomenclature issues, please see Point 4.
References
- Gu, X.; Mao, Z.; Yu, H.; Zhang, Ying.; Jiang, W.; Ling, J.; Huang, S.; Xie, B. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genomics 2011, 12(1), 471.
- Wang, L.; Zhu, W.; Fang, L.; Sun, X.; Su, L.; Liang, Z.; Wang, N.; Londo, J.P.; Li, S.; Xin, H. Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera. BMC Plant Biol. 2014, 14, 103.
- Ning, P.; Liu, C.C.; Kang, J.Q.; Lv, J.Y. Genome-wide analysis of WRKY transcription factors in wheat (Triticum aestivum L.) and differential expression under water deficit condition. PeerJ 2017, 5, 17.
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Round 2
Reviewer 3 Report
The methodology used on the determination of WRKY TFs and their nomenclature has serious flaws. The authors should use sequence identity for homology assessment, directly comparing with the AtWRKYs. The present nomenclature of the AnWRKYs is not correct at all. They should rename most of their AnWRKYs.
Author Response
Response to Reviewer 3 Comments
Point 1: The methodology used on the determination of WRKY TFs and their nomenclature has serious flaws. The authors should use sequence identity for homology assessment, directly comparing with the AtWRKYs. The present nomenclature of the AnWRKYs is not correct at all. They should rename most of their AnWRKYs.
Response 1: According to your comments, we directly compared the homology between AnWRKY and AtWRKY proteins. Based on this result, all the AnWRKY genes were renamed. Certainly, the corresponding changes in this manuscript including all the text, tables, figures and supplementary materials were made.
