BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis
, Lihua Zhong 2,3,†, Xinmin Huang 2,4, Wei Su 2, Houcheng Liu 2, Guangwen Sun 2, Shiwei Song 2,* and Riyuan Chen 2,*Round 1
Reviewer 1 Report
01st December, 2022
The manuscript ID 2081211 “BcAMT1;5 Mediates Nitrogen Influxes in Flowering Chinese Cabbage and Improves Plant Growth when Overexpressed in Arabidopsis” presents an interesting topic. The paper is well written, and I have some minor suggestions for revision.
- - Lines 81 – 85: Here, just before the objectives statement, there should be a concise rationale statement (Why this specific study was needed?). What specific knowledge gap existed?
- - Line 98 - 99: Was there variability in flowering? If there was no variability then the authors should specify the exact number of days instead of 40 – 50 days. Why were the samples collected at the flowering stage?
- - Line 192: Why are the BcAMT1;5 primers used called special BcAMT1;5 primers.
- - Lines 193-194: What was the basis for selection of the three lines used in the experiment of growth phenotyping, and the one line used for ion fluxes, N content, accumulation, and gene expression?
- - Line 238: Figure 1 has poor quality of the text within the image. The authors to replace the figure with one of higher resolution and readable text.
- Lines 437, 439, 442: the figures and tables should not be cited in the discussion. Revise appropriately.
- - Several sections (for example lines 457 – 459, 464 – 465 etc.) unnecessarily re-state the results, rather than focus on discussing the findings.
- Discussion, lines 513 - 538: the authors have been emphasizing more on your agreement with other studies than on the novelty of your own findings. So, what new have you discovered then? You are basically confirming what others have already found.
- Line 552: Change “will be” to “need to be”
Author Response
1. Lines 81 – 85: Here, just before the objectives statement, there should be a concise rationale statement (Why this specific study was needed?). What specific knowledge gap existed?
Response: Thanks for the reviewer’s good suggestion. In the revised manuscript, we have added the rationale statement that “Previous study has reported that AtAMT1;1–AtAMT1;3 mediate mainly high-affinity NH4+, AtAMT1;5 is responsible for approximately the uptake of 10% NH4+ [8]. So, most studies on AMTs are concerned with AMT1;1–AMT1;3, and very little information concerning the function of AMT1;5 exists.”. Please kindly find them in lines 86-88 of the revised manuscript.
2. Line 98 - 99: Was there variability in flowering? If there was no variability then the authors should specify the exact number of days instead of 40 – 50 days. Why were the samples collected at the flowering stage?
Response: Thank you for pointing this out. The flowering period of flowering Chinese cabbage is affected by cultivar, temperature, light, nutrient, and other factors. The seedlings of cultivar ‘Youlv 501’ are about 40-50 days from transplantation to flower at 25-30 ℃. In the present experiment, it was 48 days after transplanting for 48 days, the seedlings grew to the flowering stage. We have changed ’40-50 d’ as ’48 d’. Please kindly find them in line 106 of the revised manuscript.
As a variety of Chinese cabbage, flowering Chinese cabbage has different production organs, which include not only leaves, but also include stalks. The stalks are the stems with flower buds, and formed at the flowering stage of flowering Chinese cabbage. So, the roots, functional leaves, young leaves, stalks, petioles, and flowers were collected to analyze the expression of BcAMT1;5.
3. Line 192: Why are the BcAMT1;5 primers used called special BcAMT1;5 primers.
Response: Thank you for pointing this out. The BcAMT1;5 primers should be specific BcAMT1;5 primers. We have revised them in line 228 of the revised manuscript. Please kindly find them in line 228 of the revised manuscript.
4. Lines 193-194: What was the basis for selection of the three lines used in the experiment of growth phenotyping, and the one line used for ion fluxes, N content, accumulation, and gene expression?
Response: Thank you for pointing this out. In the experiment of growth phenotyping, the three lines of overexpression BcAMT1;5 were used to observer whether the seedlings phenotyping were consistent among different lines. According to the growth test, the three lines showed the similar phenotyping, so we selected the medium growth line ox-6 to further measure other indexes, such as ion fluxes, N content, accumulation, and gene expression. Please kindly find them in lines 228-232 of the revised manuscript.
5. Line 238: Figure 1 has poor quality of the text within the image. The authors to replace the figure with one of higher resolution and readable text.
Response: Thank you for pointing this out. We have replaced the figure with the higher resolution figure. Please kindly find them in line 281 of the revised manuscript.
6. Lines 437, 439, 442: the figures and tables should not be cited in the discussion. Revise appropriately.
Response: Thank you for pointing this out. In the revised manuscript, we have deleted the citation of the figures and tables in the discussion section.
7. Several sections (for example lines 457 – 459, 464 – 465 etc.) unnecessarily re-state the results, rather than focus on discussing the findings.
Response: Thank you for pointing this out and the reviewer’s good suggestion. In the revised manuscript, we have rewritten the related results of the discussion section. Please kindly find them in lines 524-532 and 550-554 of the revised manuscript.
8. Discussion, lines 513 - 538: the authors have been emphasizing more on your agreement with other studies than on the novelty of your own findings. So, what new have you discovered then? You are basically confirming what others have already found.
Response: Thank you for pointing this out. We have rewritten this section of discussion. In revised manuscript, we have emphasized the novel function of BcAMT1;5 from flowering Chinese cabbage. Please kindly find them in lines 586-602 of the revised manuscript.
9. Line 552: Change “will be” to “need to be”
Response: Thank you for pointing this out. We have changed “will be” to “need to be”. Please kindly find them in line 632 of the revised manuscript.
Reviewer 2 Report
The manuscript performed important results for the scientific community. The article presented a good structure and writings, updated citations and excellent discussion of the results.
Comments for author File: 
Comments.pdf
Author Response
1. In the sentence ‘BcAMT1;5 was isolated from flowering Chinese cabbage (Brassica cam-83 pestris L. ssp. chinensis var. utilis Tsen et Lee), and its expression characteristics and functions were analyzed.’ , the verb should be to conjugate in the future.
Response: Thank you for pointing this out. We have changed the verbs of the sentence as ‘BcAMT1;5, will be isolated from flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee), and its expression characteristics and functions will be analyzed’. Please kindly find them in lines 90-92 of the revised manuscript.
2. The sentence ‘kindly provided by Dr. Guibing Hu, South China Agricultural University, China’. Suggestion: quote the article.
Response: Thank you for pointing this out and for giving good suggestion. It is a great pity that we did not find the related literature to introduce this vector in the papers published by Dr. Guibing Hu's group. According to the suggestion, we have deleted the sentence ‘kindly provided by Dr. Bruno André (Université Libre de Bruxelles, Belgium)’, have added the reference ‘Marini, A.; Springael, J.; Frommer, W.B.; André, B. Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein. Mol. Microbiol. 2000, 35, 378–385. https://doi.org/10.1046/j.1365-2958.2000.01704.x.’, its order is reference [20]. Please kindly find them in lines 182-184 and 708-724 of the revised manuscript.
3. A: The phylogenetic tree of AMT family proteins was constructed using the Neighbor-Joining method in MEGA 6.0. Bootstrap 241 values were from 1000 replications, the numbers at the nodes are bootstrap values. Accession num-242 bers of AMTs protein were listed in Table S2. The statistical analysis was not mentioned in the material and methods.
Response: Thank you for pointing this out. We have added the related information in the material and methods, please kindly find them in lines 138-151 of the revised manuscript. In addition, we have modified the legend of Figure 1, please kindly find them in lines 282-299 of the revised manuscript.
Reviewer 3 Report
In this article Zhu et al., have characterized the AMT1;5 gene in Chinese cabbage using molecular approaches. The authors have shown clearly the spatial localization of AMT1;5 and change in localization under different nitrate response.
I have few comments that could help strengthen the manuscript
Major comments:
1. Although the authors have shown phenotypic evidence for AMT1;5 role, the clear data on nitrogen flux is missing. So please change the title to reflect the data that supports it. There is no in plant or heterologous transport data to support the claim.
2. Fig 2. The localization of AMT1;5 in the epidermal cell is not clear as the control image seems blurred and looks similar to AMT1;5 expression. If the authors can show proper image in tobacco or Arabidopsis protoplast with colocalization with propidium iodide it will help. Also please share quantification of fluorescence signal across the membrane using ImageJ
3. Fig8. Please explain how these images were taken and how scale bars were plotted. Were they taken with a camera? If so please mention the magnification and light settings and if any modification to image post acquisition
4. Fig 9b, the letters for statistical analysis is confusing. Please make it clear if the comparison is between roots and shoots alone or if it’s between all samples.
5. Fig 10 A & B, please add mock treated seedling image as well as data in the graph. Currently its showing only treatment.
Minor comments:
1. Line 222, phylogentic tree analysis performed here is unlikely to explain the molecular components. Please change statement.
2. Line 246 CLUSTALW spell check,
3. Line 263, please rephrase statement to reflect the experiment performed.
4. Please rewrite the conclusion section to reflect what has been shown in this paper and potential hypothesis since they are very broad claims which is not supported clearly.
Author Response
Major comments:
1. Although the authors have shown phenotypic evidence for AMT1;5 role, the clear data on nitrogen flux is missing. So please change the title to reflect the data that supports it. There is no in plant or heterologous transport data to support the claim.
Response: Thank you for pointing this out. In the revised manuscript, we have changed the title of the manuscript as “BcAMT1;5 Mediates Nitrogen Uptake and Assimilation of Flowering Chinese Cabbage and Improves Plant Growth when Overexpressed in Arabidopsis”. Please kindly find them in lines 2-4 of the revised manuscript.
2. Fig 2. The localization of AMT1;5 in the epidermal cell is not clear as the control image seems blurred and looks similar to AMT1;5 expression. If the authors can show proper image in tobacco or Arabidopsis protoplast with colocalization with propidium iodide it will help. Also please share quantification of fluorescence signal across the membrane using ImageJ
Response: Thank you for pointing this out and giving good suggestions. Actually, the image in tobacco or Arabidopsis protoplast with colocalization with propidium iodide will better response to the location of protein, we will use this colocalization test in the next study.
In the figure 2, AMT1;5 was only located in the plasma membrane of onion epidermal cell, while the control was located in the plasma membrane and cell nucleus of onion epidermal cell, so the location of AMT1;5 was different from that of the control. Since the skin of the onion is torn off with some flesh, the epidermis cells may be made up of one or several layers of cells. When taken with a fluorescence microscope, it is not easy to focus, the image of the picture will seem blurred. The ones of the control are such a case. However, the location of the control is included cell nuclear, the cell nuclear of epidermis cells must be observed, so the image of the control was used in the manuscript.
In the revised manuscript, we have added the quantification of fluorescence signal across the membrane of AMT1;5 and the control. Please kindly find them in lines 304-307 of the revised manuscript and the figure S1 of the supplementary materials.
3. Please explain how these images were taken and how scale bars were plotted. Were they taken with a camera? If so please mention the magnification and light settings and if any modification to image post acquisition
Response: Thank you for pointing this out. The images of Figure 8 were taken by the Camera Control Pro 2, which can remotely control most functions of Nikon digital camera from a computer and a digital stereo microscope connecting via USB cable. At the same magnification time of microscope, the images of ruler were taken; the scale bars were marked by photoshop according to the images of ruler.
All the images of Figure 8, were taken by the Camera Control Pro 2 software, which can remotely control most functions of Nikon digital camera from a computer and a digital stereo microscope connecting via USB cable. In the windows of “Live View”, all the images were taken at the same magnification time and shot by autofocus before each shot.
4. Fig 9b, the letters for statistical analysis is confusing. Please make it clear if the comparison is between roots and shoots alone or if it’s between all samples.
Response: Thank you for pointing this out. In Figure 9b, the letters for statistical analysis were compared among the roots or shoots of wild-type and the ox lines alone. We have graphed the figure 9b, the fresh weight of roots of wild-type and the ox lines were put together, and the ones of shoots were get together. Please kindly find them in new Figure 9b of the revised manuscript.
5. Fig 10 A & B, please add mock treated seedling image as well as data in the graph. Currently its showing only treatment.
Response: Thank you for pointing this out. In Figure 10A &B, the images and data of seedlings treated by 20 mmol L-1 MeA were shown. The mock seedlings were grown under 0.25 mmol L-1 NH4+, the images and data were shown in Figure 9, so the ones of mock were not shown in Figure 10A &B.
Minor comments:
1. Line 222, phylogentic tree analysis performed here is unlikely to explain the molecular components. Please change statement.
Response: Thank you for pointing this out. We have changed “the molecular components” as “biological characteristics”. Please kindly find them in line 245 of the revised manuscript.
2. Line 246 CLUSTALW spell check,
Response: Thank you for pointing this out. We have revised the word “CLUSTALW”. Please kindly find it in line 290 of the revised manuscript.
3. Line 263, please rephrase statement to reflect the experiment performed.
Response: Thank you for pointing this out. We have revised the statement of yeast functional complementation analysis. Please kindly find them in lines 314-318 of the revised manuscript.
4. Please rewrite the conclusion section to reflect what has been shown in this paper and potential hypothesis since they are very broad claims which is not supported clearly.
Response: Thank you for pointing this out. We have revised the conclusion to better reflect the main content of the manuscript. Please kindly find them in lines 617-632 of the revised manuscript.
Reviewer 4 Report
Overall, this is an excellent work featuring a thoughtfull design and careful implementation of the experiments along with comprehensive method set anc conclusive results. Ironically, the only note I have is that this work is completely out of scope of the journal. However, I leave it at the discretion of the Academic Editor.
Author Response
Overall, this is an excellent work featuring a thoughtful design and careful implementation of the experiments along with comprehensive method set anc conclusive results. Ironically, the only note I have is that this work is completely out of scope of the journal. However, I leave it at the discretion of the Academic Editor.
Response: Thank you for giving good review. According to the thoughtful suggestions of the reviewers, we have modified carefully the manuscript. We trust that the revised manuscript will be suitable for the publication in the journal.
