Identifying a Detoxifying Uridine Diphosphate Glucosyltransferase (UGT), MdUGT83K2, Which Can Glycosylate the Aryloxyphenoxypropionate Herbicide
Round 1
Reviewer 1 Report
It is neccesary to clarify what mean some lettes like QPP
Author Response
Response to Reviewer 1 Comments
Point 1: It is neccesary to clarify what mean some lettes like QPP.
Response 1: Thank you very much for your suggestion, QPP is an ACCase inhibition-based herbicide, see Figure 1 for the structure, which was synthesized by our team (Wang et al., 2022). Thank you again!
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors found one novel apple glycosyltransferase MdUGT83L3 could transfer glucose to herbicide QPP in vitro and in vivo, which may be involved in plant detoxification caused by herbicide, and is useful to corp herbicide-resistance molecular breeding. This finding is meaningful for the agronomy and I thus recommend publication of this ms.
There are some revisions should be done as well:
1 The illustration of 1 & 2 should be list in Fig4&6.
2 The title of result 1 "MdUGT83K2 candidate gene identification" change to "Candidate glycosyltransferase identification" ??
3 The logic and meaning of line111 should be rewritten
4 line147, do you mean form the published microarray data we found that the apple glycosyltransferase gene family Group I may be involved in response to herbicides? The original sentence lack subject.
Author Response
Response to Reviewer 2 Comments
Point 1: The illustration of 1 & 2 should be list in Fig4&6
Response 1: Thank you very much for your suggestion. 1, QPP glycosides; 2, QPP in Figure 4; 1, QPP glycosides; 2, QPP; 3, Internal reference "IBA" in Figure 6. Already added in the legend of Figure 4 and Figure 6 respectively. Thank you again!
Point 2: The title of result 1 "MdUGT83K2 candidate gene identification" change to "Candidateglycosyltransferase identification" ??
Response 2: Thank you very much for your advice, has been modified in accordance with the requirements. Thank you again!
Point 3: The logic and meaning of line111 should be rewritten
Response 3: Thank you very much for your suggestions, we have rewritten line 111 by red mark. Thank you again!
Point 4: line147. do you mean form the published microarray data we found that the appleglycosyltransferase gene family Group I may be involved in response to herbicides? The originalsentence lack subject.
Response 4: Thank you very much for your suggestion. The original text has been revised by changing “In this study, the published microarray data found that the apple glycosyltransferase gene family Group I may be involved in response to herbicides.” to “In this study, we first speculated that Group I in the apple glycosyltransferase family may predicted function like UGT83A1 to have possible detoxification effects according to gene chip data published online [14].” Thank you again!
Author Response File: Author Response.docx
Reviewer 3 Report
This manuscript describes about the function of MdUGT83K2 in detoxifying the aryloxy-3 phenoxy propionate herbicide.
Identifying the novel resources to improve herbicide resistance has relevance, however the data is not convincing that UGT protein is providing sufficient level of tolerance or not.
There are many fundamental problems in the manuscript. The data did not present well and not convincing. It is very difficult understand the results. There is no methods mentioned how they identified the nine glycosyltransferase genes.
Figure 2. it is not clear that which housekeeping gene used and what they meant by relative expression?. It is also not clear that what age plants the QPP is applied and why they have selected those time points. Same question applies to Figure 3, what stage of plant the tissues was collected.
For figure 4. It is not clear that how the protein was made, it appears that they used GST fusion construct and which is difficult to identify by general readers. It is not clear that whether the protein used in pure or not. Authors should provide gel blots and their size exclusion data. Y- axis units are missing, what is 1, 2 peaks are not described in legends. Authors should have used UGT general target as standard to show the glycosylation and then further used QPP.
In figure 6. What are three peaks, before HPLC analysis, whether the plants could survive on QPP treatment or not otherwise it is difficult to justify weather this protein has detoxifying ability or not.
Apart from qualitative data, authors should also show quantitative conversion of QPP to confirm that this protein has detoxifying ability.
There are many minor errors in the text which needs to be taken care while presenting their results.
for examples
Page 1, Line 11 - have detoxifying function, its not clear what is it detoxifying
Line 12 – How a Chip data can show detoxifying activity?
Line 14 – What is QPP, in whole manuscript it is not expanded.
Author Response
Response to Reviewer 3 Comments
Point 1: Dentifying the novel resources to improve herbicide resistance has relevance, however the datais not convincing that UGT protein is providing sufficient level of tolerance or not.
Response 1: Thank you very much for your valuable suggestions. In this study, to illustrate the detoxification effect of MdUGT83K2, first, we demonstrated that MdUGT83K2 can glycosylate and modify QPP by in vitro enzymatic reaction; then, we obtained apple histoculture seedlings overexpressing the gene MdUGT83K2 and extracted QPP glycosides after exogenous QPP treatment to verify that MdUGT83K2 can also glycosylate and modify QPP in plants. Thank you again!
Point 2: Here are many fundamental problems in the manuscript. The data did not present well and noiconvincing.
Response 2: Thank you very much for your valuable suggestions. In this overhaul, we have carefully verified all the data and modified the data that were not presented clearly. Thank you again!
Point 3: It is very difficult understand the results. There is no methods mentioned how they identified the nine glycosyltransferase genes.
Response 3: Thank you very much for your valuable suggestion, we have introduced the reference [13] after "a phylogenetic tree of apple glycosyltransferases" in the result "2.1", i.e. Li et al., 2022. In the published paper, the evolutionary tree of apple glycosyltransferase family has been successfully constructed, in which there are seven glycosyltransferase genes in Group I, namely MdUGT83L3 (MD09G1064900), MdUGT83L4 (MD09G1064700), MdUGT83L5 (MD17G1058200), MdUGT83L6 (MD17G1058400), MdUGT83L7 (MD17G1058100), MdUGT83L8 (MD09G1065000) and MdUGT83K2 (MD09G1065400); reviewing the microarray data published in NCBI, these seven genes are predicted like UGT83A1 to have possible detoxification effects (Reference [14] ). Therefore, we treated apple seedlings with compound QPP, Real-time PCR was performed to detect the expression of these seven genes. The results revealed that MdUGT83K2 induced the most significant up-regulated expression. Thank you again!
Point 4: Figure 2. it is not clear that which housekeeping gene used and what they meant by relative expression?
Response 4: Thank you very much for your valuable suggestions, the housekeeping gene we used in this study is MdEF-1a, and its primers are qF: AACTGGTCTGACTACTGA; qR: TGACAGCAACATTCTTAAC, which we have supplemented in Table S1; the relative expression in the Real-time PCR quantitative method is achieved by the relative expression in the Real-time PCR quantification method is quantified by detecting the change in expression of the target gene relative to the internal reference gene, which allows comparison of the difference in expression of the target gene between samples of different sources. Thank you again for your valuable suggestions.
Point 5: lt is also not clear that what age plants the QPP is applied and why they haveselected those time points.
Response 5: Thank you very much for your valuable suggestions, the seedlings we used for QPP treatment in this study are normal cultivated for 3 weeks ‘gala’, the seedlings at this stage have 6-8 leaves, good leaf absorption, basically stable growth characteristics, and the strain is not too big, which is very suitable for exogenous spraying trials. Thank you again.
Point 6: Same question applies to Figure 3, what stage of plant the tissues was collected.
Response 6: Thank you very much for your valuable suggestions. The materials for the spatio-temporal expression pattern of MdUGT83K2 in this study were obtained from the roots, stems, leaves, flowers and fruits of 10-year-old "Gala" apple trees grown in the College of Life Sciences of Shandong Agricultural University, as described in the Materials and Methods section "5.1 Plant material " in the Materials and Methods section. Thank you again!
Point 7: For fiqure 4. lt is not clear that how the protein was made, it appears that they used GST fusionconstruct and which is difficult to identify by general readers.
Response 7: Thank you very much for your valuable suggestions. To obtain the exogenously expressed protein of MdUGT83K2, we first cloned MdUGT83K2, followed by ligating it to the PGEX vector containing GST sequence and transforming it into BL21 E. coli. The MdUGT83K2-GST fusion protein was expressed by IPTG induction. Since GST is a commercial tagged protein, it can be purified by GST-tagged antibody, which in turn leads to purified GST and MdUGT83K2-GST. GST is a small peptide and is used as a control when enzymatic reaction is performed in vitro; MdUGT83K2-GST is a fusion protein and is used as an experimental group. To facilitate the reader's understanding, the Materials and Methods section "5.4 Vector construction" is supplemented with "The The correctly validated recombinant plasmid MdUGT83K2-PGEX was transformed into E. coli BL-21 receptor state, selected 3-5 picked single bacteria were inoculated into LB liquid medium (Amp+) and incubated overnight at 37℃ in a 225 rpm/min shaker. Thank you again.
Point 8: lt is not clear that whether the protein used in pure or not.
Response 8: Thank you very much for your advice, the proteins used in this study are all purified proteins, as explained as above “7”, thank you again!
Point 9: Authors should provide gel blots and their size exclusion data.
Response 9: Thank you very much for your suggestion, the protein blot map has been provided in this study, see details in Figure S1, thank you again!
Point 10: Y- axisunits are missing, what is 1, 2 peaks are not described in legends.
Response 10: Thank you very much for your suggestion, the Y-axis is the UV spectrum, indicated by uV, no specific value is marked because it is a qualitative study; 1 refers to QPP glycosides and 2 refers to QPP, which has been added in Figure 4 legends, thank you again!
Point 11: Authors should have used UGT general target as standard to show the glycosylation and then further used QPP in Figure 6.
Response 11: Thank you very much for your suggestion. To further show that MdUGT83K2 can glycosylate QPP in plants, we obtained MdUGT83K2 overexpression strains OE6 and OE11 after overexpressing the gene MdUGT83K2 into apple, and after treating wild-type and overexpression apple seedlings with QPP exogenously, QPP glycosides were extracted from each strain. The results showed that the QPP glycoside content in OE6 and OE11 was significantly higher than that of the wild type, and this result indicates that MdUGT83K2 can also glycosylate QPP in apples. Thank you again!
Point 12: What are three peaks, before HPLC analysis, whether the plants could survive on QPP treatment or not otherwise it is difficult to justify weather this protein has detoxifying ability or not.
Response 12: Thank you very much for your suggestions, 1, QPP glycosides; 2, QPP; 3, Internal reference "IBA", have been added in Figure 6 legends; In a previous study (Wang et al., 2022), we have shown that QPP acts on monocotyledons and is not toxic to dicotyledons. Therefore, in search of glycosyltransferases with detoxifying properties, we acted on dicotyledonous apples. Thank you again!
Point 13: Apart from qualitative data, authors should also show quantitative conversion of QPP to confirmthat this protein has detoxifying ability.
Response 13: Thank you very much for your suggestion, we have added the quantitative transformation of QPP in the original text, see the results "2.3In vitro enzymatic reaction to determine MdUGT83K2 glycosylation modification of QPP " as “The specific enzyme activity of MdUGT74BP1 to QPP was 1.83±0.32 (nkat/mg protein)”. Thank you again!
Point 14: Page 1, Line 11 - have detoxifying function, its not clear what is it detoxifying.
Response 14: Thank you very much for your suggestion. The original text has been revised by changing “In this study, we first speculated that Group I in the apple glycosyltransferase family may have detoxifying function according to gene chip data published online” to “In this study, we first speculated that Group I in the apple glycosyltransferase family may predicted function like UGT83A1 according to gene chip data published online”. Thank you again!
Point 15: Line 12 - How a Chip data can show detoxifying activity?
Response 15: Thank you very much for your suggestion. The original text has been revised by changing “In this study, we first speculated that Group I in the apple glycosyltransferase family may have detoxifying function according to gene chip data published online” to “In this study, we first speculated that Group I in the apple glycosyltransferase family may predicted function like UGT83A1 according to gene chip data published online”. Thank you again!
Point 16: Line 14 - What is QPP, in whole manuscript it is not expanded.
Response 16: Thank you very much for your suggestion, QPP is an ACCase inhibition-based herbicide, see Figure 1 for the structure, which was synthesized by our team (Wang et al., 2022). Thank you again!
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Authors have improved the manuscript. Hope my comments was helpful to them. However, the results section may be improved by providing providing information related to why they conducted the experiments, and at which stage plants they used, which will enhance readers attraction.
Author Response
Response to Reviewer 3 Comments
Point 1:Authors have improved the manuscript. Hope my comments was helpful to them. However, theresults section may be improved by providing providing information related to why theyconducted the experiments, and at which stage plants they used which will enhance readersattraction.
Response 1: Thank you very much for your valuable suggestions. In this study, we have modified the results by providing information related to why we conducted the experiments, and at which stage plants we used which will enhance reader sattraction. See the red mark in the original text for specific changes. Thank you again!
