Glucose-6-Phosphate Dehydrogenase Is Involved in the Tolerance of Soybean Seedlings to Low Nitrogen Stress
Round 1
Reviewer 1 Report
Have you done any field experiments of these soybean varieties?
Lines 1-2: This experiment has been carried out on soybean seedlings. It would be better to add this information into the title as well.
Line 19: Variety name should be mention first in the text then abbreviations
Lines 22-23: G6PDH was strongly induced at gene level under low N conditions? What that mean and how you did it?
Lines 93-95: I think there is no need to write result section here.
Lines 111-114: How many days in total you have grown the plants in this experiment? Please mention the complete information.
Lines 172-173: Please elaborate more about root traits measurement, better to write in a separate paragraph than in statistical analysis.
Lines 233-236: How many root samples were analysed to study the G6PDH, Cyt-G6PDH, Pla-G6PDH etc activities.? It should be clearly mentioned into the text and in legends also, in all the figures.
Lines 280-311: PCA gives us very important information’s about the behaviour of different cultivars in relation to different traits. Please re-write this paragraph in more informative way. Would be great if you can add the graph here?
Author Response
Dear Reviewer,
Thank you for the constructive comments for our article. We have revised the manuscript “Glucose-6-Phosphate Dehydrogenase Is Involved in the Tolerance of Soybean to Low Nitrogen Stress (agronomy-1137721)” according to the reviewers’ comments. In the point-by-point responses attached below, reviewers’ comments are in black fonts and our responses are in blue fonts.
- Have you done any field experiments of these soybean varieties?
>Answer: Thank you for your attention. In fact, the three soybean seeds were provided by Prof. Guohong Zhang (Gansu Academy of Agricultural Sciences). He has concentrated on cultivating high yield and resistance soybean varieties with field experiments. LH3 is a newly developed cultivar with higher yield compared with LD2 (Wang et al. 2019). JD19 was bred in 2003 with large seeds, lodging resistance and drought tolerant (He et al. 2016). In our study, we found the three cultivars exhibited different low-N tolerance at seedling stage, thus we chose them as experimental materials for further study.
He J, Du YL, Wang T, Turner N, Xi Y, Li FM (2016) Old and New Cultivars of Soya Bean (Glycine max L.) Subjected to Soil Drying Differ in Abscisic Acid Accumulation, Water Relations Characteristics and Yield. J Agro Crop Sci 202:372-383.
Wang LM, Zhang GH, Yang RP, Chen GR (2019) A new soybean cultivar LONGHUANG 3 (LH3). Chinese seed industry 4:80-81.
- Lines 1-2: This experiment has been carried out on soybean seedlings. It would be better to add this information into the title as well.
>Answer: Thank you very much for your advice. It has been added in the title (line 3).
- Line 19: Variety name should be mention first in the text then abbreviations.
>Answer: Thank you for your attention. It has been revised in the manuscript (line 19).
- Lines 22-23: G6PDH was strongly induced at gene level under low N conditions? What that mean and how you did it?
>Answer: I am sorry for my carelessness. This sentence is not very accurate. The correct description is that the G6PDH expression showed different degrees of change in these soybean cultivars under low-N conditions. It has been revised in the manuscript (line23-24).
- Lines 93-95: I think there is no need to write result section here.
>Answer: Thank you for your attention. It has been deleted in the manuscript.
- Lines 111-114: How many days in total you have grown the plants in this experiment? Please mention the complete information.
>Answer: Thank you for your attention. Our material treatments were divided into two parts. One part was low-N and control-N treatment, under this condition, the soybean seedlings were cultured continuously for 10 days (line103-105). The other part was exogenous inhibitor of G6PDH (GlcN) treatment, under this condition, the inhibitor and low-N or control-N were co-treated soybean seedlings for 2 d after 10 d of low-N and control-N treatment (lines 105-113).
- Lines 172-173: Please elaborate more about root traits measurement, better to write in a separate paragraph than in statistical analysis.
>Answer: Thank you for your advice. It has been separated a single paragraph (lines 118-121).
- Lines 233-236: How many root samples were analyzed to study the G6PDH, Cyt-G6PDH, Pla-G6PDH etc activities? It should be clearly mentioned into the text and in legends also, in all the figures.
>Answer: Thank you for your attention. 0.5 g of roots and leaves were used for determination of G6PDH activity. And each assay was carried out with three independent biological replicates. It has been added in the manuscript (line 182).
- Lines 280-311: PCA gives us very important information’s about the behaviour of different cultivars in relation to different traits. Please re-write this paragraph in more informative way. Would be great if you can add the graph here?
>Answer: Thank you for your advice. We have been re-wrote this paragraph and added a graph in the manuscript (Supplementary Table S2).
Reviewer 2 Report
The article describing the role of G6PDH is interesting. I have a major concern for the genotyping variation observed under low nitrogen stress. The observed genotypic variation looks uncertain. Even in soybean, the role of G6PDH is highly studied. Authors can find my publications for G6PDH role under stress. Therefore, it is required to build upon the available knowledge with well-planned experiments. No doubt the present study is well-planned and the results are also interesting, but the use of only three genotypes is not sufficient to conclude on genotypic variation. I suggest including at least 20-25 diverse genotypes.
In the introduction, the authors have correctly stated that the "Previous studies mostly focused on the relationship between G6PDH and salt or drought stress." Therefore the mechanism under these trait compared to low nitrogen stress need to be discussed. The physiological processes are common for different stress.
Materials and methods
"In each independent assay, 0.5 g of samples were used in each single repeat." - leaf, root or seeds?
Please add a brief method for GHS quantification.
How many biological replications were considered for each assay.
Author Response
Dear Reviewer,
Thank you for the constructive comments for our article. We have revised the manuscript “Glucose-6-Phosphate Dehydrogenase Is Involved in the Tolerance of Soybean to Low Nitrogen Stress (agronomy-1137721)” according to the reviewers’ comments. In the point-by-point responses attached below, reviewers’ comments are in black fonts and our responses are in blue fonts.
- The article describing the role of G6PDH is interesting. I have a major concern for the genotyping variation observed under low nitrogen stress. The observed genotypic variation looks uncertain. Even in soybean, the role of G6PDH is highly studied. Authors can find my publications for G6PDH role under stress. Therefore, it is required to build upon the available knowledge with well-planned experiments. No doubt the present study is well-planned and the results are also interesting, but the use of only three genotypes is not sufficient to conclude on genotypic variation. I suggest including at least 20-25 diverse genotypes.
>Answer: Thank you very much for your advice. In fact, the three soybean genotypes have long-term field experiment data and its own characteristics. JD19 was bred in 2003 with large seeds, lodging resistance and drought tolerant (He et al. 2016). LH3 is a newly developed cultivar with high yield and good quality. LD2 was bred in 2005, with 4-5 branches and small seeds (Wang et al. 2019). In our study, we found the three cultivars exhibited different low-N tolerance, thus, we chose them as experimental materials. In next step, we will definitely look for more genotypes for further study.
He J, Du YL, Wang T, Turner N, Xi Y, Li FM (2016) Old and New Cultivars of Soya Bean (Glycine max L.) Subjected to Soil Drying Differ in Abscisic Acid Accumulation, Water Relations Characteristics and Yield. J Agro Crop Sci 202:372-383.
Wang LM, Zhang GH, Yang RP, Chen GR (2019) A new soybean cultivar LONGHUANG 3 (LH3). Chinese seed industry 4:80-81.
- In the introduction, the authors have correctly stated that the "Previous studies mostly focused on the relationship between G6PDH and salt or drought stress." Therefore the mechanism under these trait compared to low nitrogen stress need to be discussed. The physiological processes are common for different stress.
>Answer: Thank you very much for your advice. It has been discussed in the manuscript (lines 397-406).
- Materials and methods - "In each independent assay, 0.5 g of samples were used in each single repeat." - leaf, root or seeds?
>Answer: Thank you for your attention. 0.5 g of samples means 0.5 g leaves and 0.5 g roots. It has been revised in the manuscript (line130).
- Materials and methods - Please add a brief method for GSH quantification.
>Answer: Thank you for your attention. It has been added in the manuscript (lines 135-137).
- Materials and methods - How many biological replications were considered for each assay.
>Answer: Thank you for your attention. Each experiment was carried out with at least three biological replicates. It has been added in the manuscript (line 182).
