The Effect of Nitrogen Fertilizer on Rhizome Quality and Starch Physicochemical Properties in Nelumbo nucifera
Round 1
Reviewer 1 Report
I appreciate the level of paper from all aspects. It is clearly written for potential readers /researchers.
Author Response
Thank you very much for your comments and suggestions. We have checked the manuscript and revised the language and style.
Reviewer 2 Report
In this study, the authors performed a comparative analysis to understand the effect of nitrogen fertilizer on the development of lotus (Nelumbo nucifera) at physiological and transcriptional levels. The rhizome of the lotus is a popular aquatic vegetable with economic values, thus the aims and the topic have practical implications in agricultural production. They succeeded in identifying the most effective nitrogen concentration (N2) by concentration gradient experiment. Their results of physiological experiments and raw data of transcriptome sequencing would be helpful for further investigation in the lotus. However, there are some errors and doubts in the current version, including the following points.
Major:
(1) Lines 33-35: According to the latest phylogenetic tree analysis, the Nelumbo nucifera Geatn. belongs to the genus Nelumbo in the family Nelumbonaceae (DOI: 10.1186/s12870-014-0289-0, 10.1093/molbev/msaa105). A manifest error at the beginning of the Introduction would greatly reduce the professionalism and quality of the article. Meanwhile, the three types of lotus (rhizome, flower, and seed lotus) have different phenotypes but shared the same biological properties. And the differential phenotypes are the results of different directions of artificial cultivation and breeding.
(2)Line 84-87: Only the nitrogen concentrations were presented, the process of nitrogen fertilizer was not mentioned. As the authors mentioned in the Introduction, N-fertilizer is hydrosoluble and would pollute the water environment. They plant the lotus in a plot, but the method by which N-fertilizer was applied in the water or under the soil was unclear. If N-fertilizer was applied in the water, it would be a direct source of pollution. Please explain it carefully.
(3) Line 129: The authors carried out RNA-seq for the rhizome of the lotus in different nitrogen fertilizer treatments. However, their pipeline of transcriptome analysis lacks a high-quality reference genome. Because previous studies have assembled several reference genomes in wild lotus species and Li et al.(2021) published a comprehensive Nelumbo Genome Database (http://nelumbo.biocloud.net/nelumbo/home), the authors should have a reference genome when they run the pipeline of transcriptome analysis. Although they used Trinity to de novo assembly unigenes, the short reads from Next-generation sequencing are week to obtain full-length transcripts and unigenes with complete ORF. Please reanalyzed the transcriptome data with a reference genome.
(4) Line 234: A total of 12 RNA libraries were sequenced in the M&M. But the differential expression analysis was carried out based on reads from only 11 libraries. Here is a writing mistake or the authors abandon one.
(5) The hypothetical test types were missed in the whole manuscript. When comparing the results, the authors highlighted that the difference was significant and shown in different letters, but the type of hypothetical test was largely unclear. Please add it to the good places.
(6) The pixel in all figures was too low, please improve the quality of all figures.
Minor:
Line 37: which part of the lotus has medicinal values, please add the relevant cites.
Line 40: Did the author count the cultivated area of the lotus? If not, please add the cite.
Line 153,159: “p-value” and “p value”, please unify them in the manuscript.
Line 160: The “Nelumbo nucifera Genome” or “Kyoto Encyclopedia of Genes and Genomes”
Line 217: The quality of the rhizome is ambiguous, please define a specific character of lotus rhizome that was investigated by the authors.
Author Response
(1) Lines 33-35: According to the latest phylogenetic tree analysis, the Nelumbo nucifera Geatn. belongs to the genus Nelumbo in the family Nelumbonaceae (DOI: 10.1186/s12870-014-0289-0, 10.1093/molbev/msaa105). A manifest error at the beginning of the Introduction would greatly reduce the professionalism and quality of the article. Meanwhile, the three types of lotus (rhizome, flower, and seed lotus) have different phenotypes but shared the same biological properties. And the differential phenotypes are the results of different directions of artificial cultivation and breeding.
Response 1: Thank you very much for your comments and suggestions. We realized the seriousness of the manifest error and have revised them in the manuscript. Nelumbo nucifera Gaertn is a perennial aquatic vegetable of the genus Nelumbo in the family Nelumbonaceae. And we agree with your comments it is more accurate to classify Nelumbo nucifera Gaertn based on different phenotypes.
(2) Line 84-87: Only the nitrogen concentrations were presented, the process of nitrogen fertilizer was not mentioned. As the authors mentioned in the Introduction, N-fertilizer is hydrosoluble and would pollute the water environment. They plant the lotus in a plot, but the method by which N-fertilizer was applied in the water or under the soil was unclear. If N-fertilizer was applied in the water, it would be a direct source of pollution. Please explain it carefully.
Response 2: Thank you very much to point out the detail. Nitrogen (N) is a major limiting nutrient for plant growth and development, especially for crops in the agroecosystems. N-fertilizer is hydrosoluble and would pollute the water environment. To decrease the waste of nitrogen and pollution for water, we conducted the mixture (wet soil : nitrogen=2:1) and then spread evenly over the plot. In this way, the nitrogen could sink directly to the bottom of the plot water. We added the detailed applied method of nitrogen fertilizer in the part of method.
(3) Line 129: The authors carried out RNA-seq for the rhizome of the lotus in different nitrogen fertilizer treatments. However, their pipeline of transcriptome analysis lacks a high-quality reference genome. Because previous studies have assembled several reference genomes in wild lotus species and Li et al.(2021) published a comprehensive Nelumbo Genome Database (http://nelumbo.biocloud.net/nelumbo/home), the authors should have a reference genome when they run the pipeline of transcriptome analysis. Although they used Trinity to de novo assembly unigenes, the short reads from Next-generation sequencing are week to obtain full-length transcripts and unigenes with complete ORF. Please reanalyzed the transcriptome data with a reference genome.
Response 3: Thank you very much for your suggestions. A high-quality reference genome is very important for the analysis of RNA-seq. And now there are three published Nelumbo nucifera Gaertn genome, containing wild strain of sacred lotus (2013), the sacred lotus variety ‘China Antique’ (2013), Nelumbo Adans. ‘Taikonglian No.3’ (2022). In fact, we have sequenced the genome of the main cultivar of lotus ‘MRH’ cultured in Jiangsu province by Third-generation Sequencing. The data have been analyzing and not been published. Therefore, we could ensure the analysis reliability of transcriptome data.
(4) Line 234: A total of 12 RNA libraries were sequenced in the M&M. But the differential expression analysis was carried out based on reads from only 11 libraries. Here is a writing mistake or the authors abandon one.
Response 4: Thank you for your careful work. We are sorry that we made a mistake. We have revised the writing mistake.
(5) The hypothetical test types were missed in the whole manuscript. When comparing the results, the authors highlighted that the difference was significant and shown in different letters, but the type of hypothetical test was largely unclear. Please add it to the good places.
Response 5: Thank you very much for your suggestions. In this research, there are five concentration of nitrogen treatment, 0 kg/667 m2 (CK), 20 kg/667 m2 (N1), 30 kg/667 m2 (N2), 40 kg/667 m2 (N3) and 50 kg/667 m2 (N4). And hypothetical test was analyzed by software SPSS. Means not sharing a common letter are significantly different between groups at P = 0.05 as determined by LSD after a one-way ANOVA test. And the related information was added in the method part.
(6) The pixel in all figures was too low, please improve the quality of all figures.
Response 6: Thank you very much. We have replaced with higher pixel images which was 600 dpi and tiff image format.
(7) Line 37: which part of the lotus has medicinal values, please add the relevant cites.
Response 7: Thank you for reminding. In fact, all parts of the lotus have been used for various medicinal purposes in various systems of medicine including folk medicines, Ayurveda, Chinese traditional medicine, and oriental medicine. Many chemical constituents have been isolated till the date. (https://doi.org/10.1155/2015/789124). And we have added the relevant cites.
(8) Line 40: Did the author count the cultivated area of the lotus? If not, please add the cite.
Response 8: According to China characteristic vegetable industry technology system statistics, the planting area of lotus has reached 600,000 hectares in 2021 in China.
(9) Line 153,159: “p-value” and “p value”, please unify them in the manuscript.
Response 9: Thank you very much for your reminding. We have revised the manuscript and unified them.
(10) Line 160: The “Nelumbo nucifera Genome” or “Kyoto Encyclopedia of Genes and Genomes”
Response 10: Thank you for your careful work. We are sorry that we made a mistake and we have revised it. Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.kegg.jp/kegg/pathway.html) was the right.
(11) Line 217: The quality of the rhizome is ambiguous, please define a specific character of lotus rhizome that was investigated by the authors.
Response 11: The starch fine structure containing the number, length, size and branch degree of amylose and amylopectin, which can affect the rhizome cooking and starch pasting characteristic.
Author Response File: 
Author Response.docx
Reviewer 3 Report
The grammar and punctuation should be checked accurately.
The latin name of the plant should be italised.
Numeric indexes should be subscripted and superscripted (eg., CO2, etc).
line 85:
The the housekeeping genes and mastermix used for RT-PCR were not mentioned.
Poor quality of figures: impossible to read the text within the figures.
Figure 8. Should be checked for accuracy: few graphs without scale, or the name of the axis.
Author Response
1. The grammar and punctuation should be checked accurately. The latin name of the plant should be italised. Numeric indexes should be subscripted and superscripted (eg., CO2, etc).
Response 1: Thank you very much for your comments and suggestions. We have checked all the manuscript and revised these mistakes.
2. line 85: The housekeeping genes and mastermix used for RT-PCR were not mentioned.
Response 2: Thank you very much for your reminding. The housekeeping gene was TURA, which encode the Nelumbo nucifera tubulin alpha-3 chain. And the mastermix used for qRT-PCR was SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). We added the detailed information in the method part.
3. Poor quality of figures: impossible to read the text within the figures.
Response 3: Thank you very much. We have replaced with higher pixel images which was 300 dpi and tiff image format.
4. Figure 8. Should be checked for accuracy: few graphs without scale, or the name of the axis.
Response 4: Thank you very much for your careful work. We are sorry that we made a mistake and we have checked accurately and revised it.
