Effects of Foliar Spraying of Organic Selenium and Nano-Selenium Fertilizer on Pak Choi (Brassica chinensis var. pekinensis. cv. ‘Suzhouqing’) under Low Temperature Stress
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study demonstrates a rigorous and comprehensive approach to analyzing RNA-Seq data, and the results contribute valuable insights into the effects of selenium treatment on gene expression. The findings have potential implications for agricultural practices and warrant further discussion in the context of the broader scientific literature. The study provides a detailed and thorough analysis of RNA-Seq sequencing data obtained from control and selenium-treated groups. The methods employed, including CASAVA base recognition and Trimmomatic software, demonstrate a rigorous approach to data processing, ensuring high-quality results with minimal sequencing error rates. However, some of the following minor and major concerns need to be addressed.
Comments
1. The methodology does not explicitly mention the number of replicates for each treatment. Providing this information is crucial for assessing the statistical robustness of the results.
Moreover, statistical tests conducted for biochemical analysis such table 1. 2 and 3 should be mentioned in methodology section. The statistical methods used for data analysis need to be described more comprehensively.
Tables 1, 2 and 3 needs comprehensive description. Table 1: Note: Lowercase letters indicate significant difference between treatments (0.05) in this paper. Significant differences based on which statistical test? 0.05 indicate P value?
It would be beneficial to briefly explain the rationale behind the specific timing of selenium application on December 9th, 16th, and 27th.
The organic selenium fertilizer and nano-selenium fertilizer were applied three times…… “ how much amount of organic selenium fertilizer were applied?
It would be helpful to specify the exact forms or compounds of organic and nano-selenium used for treatment.
It is essential to explicitly clarify what the control group (LCK) represents. Does it receive a standard treatment or serve as a baseline with no selenium treatment?
The use of RNA-Seq and Weighted Gene Co-expression Network Analysis (WGCNA) is briefly mentioned, but more details about the methodologies employed would be beneficial for peer reviewers to evaluate the study's rigor.
2. While the study mentions the use of Trimmomatic software and the Q30 quality score threshold, it would be helpful to provide specific details about the parameters used in the trimming process. This ensures transparency and reproducibility.
3. The study describes the use of a hypergeometric distribution test for GO enrichment analysis. However, it would be significant to note that if any multiple testing corrections were applied?
The abstract touches on the molecular mechanisms involving photosynthetic pigments, the antioxidant system, and selenium absorption and metabolism. Providing more context on how these mechanisms contribute to low-temperature tolerance and selenium content enhancement would enhance the clarity of the abstract
4. Figure captions should be more descriptive. For example, in Figure 2, it would be beneficial to include a brief explanation of what the volcano plot represents. Moreover, Fig 6,7 and 8 requires comprehensive description.
The identification of highly correlated gene co-expression modules and the discovery of hub genes is a key finding. It would be helpful to describe the biological significance of these findings and compare with existing information in previous studies. How do these modules and hub genes relate to physiological traits and stress resistance?
Names of the genes should be mentioned in italic form throughout the manuscript.
Some sentences, such as "The seeds were sown in a culture dish and allowed to grow for one week. Once the seedlings developed three leaves and a heart-shaped structure, they were transplanted into pot," could be rephrased for greater clarity and flow. It is recommended to do proper language editing of entire manuscript.
Selenium fertilizer mainly includes inorganic selenium, organic selenium and nano- 50 selenium. Inorganic selenium fertilizers, such as sodium selenate or sodium selenite, ex- 51 hibit low absorption and utilization conversion at low concentrations and high toxicity at 52 high concentrations when applied through foliage or soil “ Provide citation”.
The Suzhouqing variety is pak choi (Brassica chinensis var. pekinensis), belonging to the ordinary cabbage type. It possesses high nutritional value and ranks among the vegetables with high content of various vitamins and mineral elements. Moreover, it can effectively maintain blood vessel elasticity over an extended period of time. Additionally, this variety is rich in fiber, which aids in alleviating constipation and provides essential nutrients for normal physiological requirements while enhancing the body's innate immunity. “Provide relevant references and citations to the above text in introduction part”.
Comments on the Quality of English LanguageThere are some minor mistakes in manuscript. Such as some sentences "The seeds were sown in a culture dish and allowed to grow for one week. Once the seedlings developed three leaves and a heart-shaped structure, they were transplanted into pot," could be rephrased for greater clarity and flow.
Author Response
Dear professor,
On behalf of co-authors, we are very grateful to you for giving us an opportunity to revise our manuscript. We appreciate you very much for your positive and constructive comments and suggestions on our manuscript. Your feedback has been extremely helpful in revising and improving our paper, as well as guiding our research.
We have carefully considered your suggestions and tried our best to revise our manuscript according to the comments. The revised portions are highlighted in red within the paper. Thanks again to the hard work of reviewer!
Response to the comments
Comment No. 1: The methodology does not explicitly mention the number of replicates for each treatment. Providing this information is crucial for assessing the statistical robustness of the results.
Response: Thanks to the reviewer for the reminder, and we have added the number of replicates for each treatment in section 2.1 (p3 LN96).
Comment No. 2: Moreover, statistical tests conducted for biochemical analysis such table 1. 2 and 3 should be mentioned in methodology section. The statistical methods used for data analysis need to be described more comprehensively.
Response: Thank you very much for your suggestion. We have added the statistical analysis in section 2.8 (p4 LN173-175).
Comment No. 3: Tables 1, 2 and 3 needs comprehensive description. Table 1: Note: Lowercase letters indicate significant difference between treatments (0.05) in this paper. Significant differences based on which statistical test? 0.05 indicate P value?
Response: Thank you very much for your suggestion. We have added p<0.05 for Table 1 (p5 LN193).
Comment No. 4: It would be beneficial to briefly explain the rationale behind the specific timing of selenium application on December 9th, 16th, and 27th.
Response: Thank you very much for your suggestion. According to the instructions of organic selenium and nano-selenium, it can be sprayed 3 times in the middle and late stage of vegetable growth, and the interval of 7-10 days is appropriate. Therefore, according to the actual situation, we chose December 9th, 16 th and 27 th to spray.
Comment No. 5: The organic selenium fertilizer and nano-selenium fertilizer were applied three times…… “ how much amount of organic selenium fertilizer were applied?
Response: Thank you very much for your question. Leaves were sprayed with 20 mL of organic selenium or nano-selenium. We have added this important information in section 2.1 (p2 LN91-94).
Comment No. 6: It would be helpful to specify the exact forms or compounds of organic and nano-selenium used for treatment.
Response: Thank you very much for your suggestion. We have added the compounds of organic selenium in section 2.1 (p2 LN95).
Comment No. 7: It is essential to explicitly clarify what the control group (LCK) represents. Does it receive a standard treatment or serve as a baseline with no selenium treatment?
Response: Thank you very much for your suggestion. The control group (LCK) was sprayed with 20 ml of water. This information was added in in section 2.1 (p2 LN91).
Comment No. 8: The use of RNA-Seq and Weighted Gene Co-expression Network Analysis (WGCNA) is briefly mentioned, but more details about the methodologies employed would be beneficial for peer reviewers to evaluate the study's rigor.
Response: Thank you very much for your suggestion. We have added the brief introduction of Weighted Gene Co-expression Network Analysis (WGCNA) in section 2.6 (p4 LN153-160).
Comment No. 9: While the study mentions the use of Trimmomatic software and the Q30 quality score threshold, it would be helpful to provide specific details about the parameters used in the trimming process. This ensures transparency and reproducibility.
Response: Thank you very much for your suggestion. We have added some specific details about the parameters used in the trimming process in section 2.4 (p3 LN121-134).
Comment No. 10: The study describes the use of a hypergeometric distribution test for GO enrichment analysis. However, it would be significant to note that if any multiple testing corrections were applied?
Response: Thank you very much for your suggestion. We have added the statistical analysis in section 2.8 (p4 LN173-175).
Comment No. 11: The abstract touches on the molecular mechanisms involving photosynthetic pigments, the antioxidant system, and selenium absorption and metabolism. Providing more context on how these mechanisms contribute to low-temperature tolerance and selenium content enhancement would enhance the clarity of the abstract
Response: Thank you very much for your suggestion. We have added the mechanisms contribute to low-temperature tolerance and selenium content enhancement in abstract (p1 LN17-24).
Comment No. 12: Figure captions should be more descriptive. For example, in Figure 2, it would be beneficial to include a brief explanation of what the volcano plot represents. Moreover, Fig 6,7 and 8 requires comprehensive description.
Response: Thank you very much for your suggestion. We have added the brief explanations for Fig. 2, 6, 7 and 8.
Comment No. 13: The identification of highly correlated gene co-expression modules and the discovery of hub genes is a key finding. It would be helpful to describe the biological significance of these findings and compare with existing information in previous studies. How do these modules and hub genes relate to physiological traits and stress resistance?
Response: Thank you very much for your suggestion. We have added the “nine hub genes identified by Weighted Gene Co-expression Network Analysis (WGCNA) were closely related to the mentioned endocytosis, antioxidant, ABC transporter and selenium metabolism, and the results of functional enrichment analysis were similar to the results of Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis by RNA-seq, thus confirming the reliability of this analysis” in conclusion (p22 LN676-680).
Comment No. 14: Names of the genes should be mentioned in italic form throughout the manuscript.
Response: Thank you very much for your suggestion. All the gene names are now in italics.
Comment No. 15: Some sentences, such as "The seeds were sown in a culture dish and allowed to grow for one week. Once the seedlings developed three leaves and a heart-shaped structure, they were transplanted into pot," could be rephrased for greater clarity and flow. It is recommended to do proper language editing of entire manuscript.
Response: Thank you very much for your suggestion. We have rewritten the plant materials and experimental conditions to make it clear (p2 LN81-99).
Comment No. 16: Selenium fertilizer mainly includes inorganic selenium, organic selenium and nano-selenium. Inorganic selenium fertilizers, such as sodium selenate or sodium selenite, exhibit low absorption and utilization conversion at low concentrations and high toxicity at high concentrations when applied through foliage or soil “ Provide citation”.
Response: Thank you very much for your suggestion. Since the objectives of this paper are organic selenium and nano-selenium, rather than inorganic selenium, we have chosen to delete the content about inorganic selenium.
Comment No. 17: The Suzhouqing variety is pak choi (Brassica chinensis var. pekinensis), belonging to the ordinary cabbage type. It possesses high nutritional value and ranks among the vegetables with high content of various vitamins and mineral elements. Moreover, it can effectively maintain blood vessel elasticity over an extended period of time. Additionally, this variety is rich in fiber, which aids in alleviating constipation and provides essential nutrients for normal physiological requirements while enhancing the body's innate immunity. “Provide relevant references and citations to the above text in introduction part”.
Response: Thank you very much for your suggestion. We have added the reference in introduction (p1 LN38-41).
Comment No. 18: There are some minor mistakes in manuscript. Such as some sentences "The seeds were sown in a culture dish and allowed to grow for one week. Once the seedlings developed three leaves and a heart-shaped structure, they were transplanted into pot," could be rephrased for greater clarity and flow.
Response: Thank you very much for your suggestion. We have rewritten the plant materials and experimental conditions to make it clear (p2 LN81-99).
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsSome information about the experiment conditions should be added to the Materials and Methods section.
insufficient description of experimental design, materials, and methods generally.
Clearly state whether your objective is novel or not.
The introduction is quite poor, and sentences sometimes seem to be conceptually disconnected from each other, overall in the final part. Due to the seriousness of the implications of such work, more care must be given to the preparation of the introduction.
The hypotheses and objectives should be clearly provided.
The Material and Methods section needs a strong revision: single paragraphs are badly structured, resulting in confusion.
insufficient presentation (remarkable comparison of means) and description of the results. There are too many illustrations, which are also insufficiently annotated.
It was often difficult to see how conclusions on results were drawn from the illustrations, whereas other effects were not presented.
The discussion is not actually a discussion but more a list of references for each measured variable. There is no actual analysis of the processes that occur in the experiments, and no practical conclusions are drawn. The discussion does not really discuss the experimental results found here but lists numerous literature references. One does not see what is new here, what is consistent with or different from previous studies, or why the present study was necessary.
The variables measured in the experiment were "discussed" without any links between them and without trying to conclude from their simultaneous measurements.
The English of the MS should be improved.
Author Response
Dear professor,
On behalf of co-authors, we are very grateful to you for giving us an opportunity to revise our manuscript. We appreciate you very much for your positive and constructive comments and suggestions on our manuscript. Your feedback has been extremely helpful in revising and improving our paper, as well as guiding our research.
We have carefully considered your suggestions and tried our best to revise our manuscript according to the comments. The revised portions are highlighted in red within the paper. Thanks again to the hard work of reviewer!
Response to the comments
Comment No. 1: Some information about the experiment conditions should be added to the Materials and Methods section, insufficient description of experimental design, materials, and methods generally.
Response: Thank you very much for your suggestion. We have rewritten the plant materials and experimental conditions and determination of physiological and biochemical indexes to make it clear (p2 LN81-112). And we added the method of RNA extraction and RNA-Seq sequencing, Identification of gene differential expression, GO and KEGG enrichment analysis of DEGs, Analysis of WGCNA, Quantitative real-time PCR and Statistical analysis in section 2 (p2 Ln113-175).
Comment No. 2: Clearly state whether your objective is novel or not. The introduction is quite poor, and sentences sometimes seem to be conceptually disconnected from each other, overall in the final part. Due to the seriousness of the implications of such work, more care must be given to the preparation of the introduction. The hypotheses and objectives should be clearly provided.
Response: Thank you very much for your suggestion. We have rewritten the introduction (p1 LN3-79): The pak choi (Brassica chinensis var. pekinensis. cv. ‘Suzhouqing’) is rich in fiber, vitamins and mineral elements. It can be planted in four seasons, so it occupies a very important position in the supply of vegetables throughout the year. Although this variety has stronger cold resistance than other varieties of Chinese cabbage, low temperature environment will reduce its yield and quality. Some results have shown that selenium fertilizer could reduce the effect of low temperature stress on crops. Therefore, this study tried to apply selenium fertilizer (organic selenium and nano-selenium) to Suzhouqing to resist low temperature stress. However, the mechanism of Suzhouqing response to nano-selenium and organic selenium under low temperature by RNA-Seq has not been reported so far. So our objective is to investigate whether organic selenium and nano-selenium can enhance low-temperature tolerance and increase selenium content in pak choi, and we think it is noval.
Comment No. 3: The Material and Methods section needs a strong revision: single paragraphs are badly structured, resulting in confusion.
Response: Thank you very much for your suggestion. We have rewritten the plant materials and experimental conditions to make it clear (p2 LN81-99).
Comment No. 4: insufficient presentation (remarkable comparison of means) and description of the results. There are too many illustrations, which are also insufficiently annotated. It was often difficult to see how conclusions on results were drawn from the illustrations, whereas other effects were not presented.
Response: Thank you very much for your suggestion. We have modified the conclusion to better explain the logic of this study (p21 LN661-680). At first, the results showed that exogenous application of selenium increased the photosynthesis of leaves, increased the activity of antioxidant enzymes, increased the weight of leaves, and increased the selenium content of leaves through the absorption and transport of selenium. The application of exogenous selenium can not only induce plant growth and nutritional quality, but also induce the expression of a series of related genes in plants, thus causing various physiological response mechanisms. So the transcriptome sequencing of leaves under different treatments was conducted by RNA-seq technology, and we systematically analyzed the specific reasons. The beneficial effects on yield and total selenium content under low temperature were attributed to (1) protection of photosynthetic pigments for enhancing photosynthetic capacity by the up-regulation of LHca2, LHcb1, LHca1, LHcb4 in KEGG pathway: photosynthesis-antenna proteins; (2) activation of antioxidant system for efficient ROS homeostasis such as SOD, POD and CAT by the genes such as Superoxide dismutase, Monodehydroascorbate and (3) selenium absorption by endocytosis, seleninum transportation by ABC transporter gene family and selenium metabolism related genes such as Cysteine synthase, Glutaredoxin. Then we used WGCNA to determin nine hub genes whic were closely related to the mentioned endocytosis, antioxidant, ABC transporter and selenium metabolism. Finally, we found that the results of functional enrichment analysis were similar to the results of GO enrichment analysis and KEGG pathway analysis by DEGs mentioned in the paper, so we confirmed the reliability of this results.
Comment No. 5: The discussion is not actually a discussion but more a list of references for each measured variable. There is no actual analysis of the processes that occur in the experiments, and no practical conclusions are drawn. The discussion does not really discuss the experimental results found here but lists numerous literature references. One does not see what is new here, what is consistent with or different from previous studies, or why the present study was necessary.
Response: Thank you very much for your suggestion. In the discussion section, we add a comparison between the results of this paper and the conclusions of others, and further analyze the reasons why the results are similar or different, shown in section 4.1-4.4 (p19 LN532-614).
Comment No. 6: The variables measured in the experiment were "discussed" without any links between them and without trying to conclude from their simultaneous measurements.
Response: Thank you very much for your suggestion. The variables measured in this article are all related. By upregating LHca2, LHcb1, LHca1 and LHcb4 proteins in KEGG pathway, photosynthetic pigments were protected and photosynthetic capacity was enhanced. At the same time, SOD, POD, CAT and other antioxidant systems were activated by genes such as superoxide dismutase and monodehydroascorbic acid to achieve efficient homeostasis of ROS, thus increasing production. Selenium was absorbed by endocytosis, transported by ABC transport gene family, and metabolized by cysteine synthetase, Glutaredoxin and other selenium metabolism-related genes, which increased selenium content.
Comment No. 7: The English of the MS should be improved.
Response: Thank you very much for your suggestion. We have taken great effort to modify the sentences to make them more professional. The portion of language modification are marked in red in the revised manuscript, and we hope it can meet with requirement.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsLN39: Selenium may be termed a 'beneficial element', instead of 'micronutrient'
LN62-63: rephrase the sentence
LN83: incomplete
LN88-91: rephrase the sentences
LN 94 and 98: check the mentioned date
LN146 and elsewhere: how do the authors justify the increase in SOD activity by 341.16%? When the percent increase is more than 100, better to express it in fold-change units.
LN178-179: "In comparison to control, GSH content sprayed with organic selenium was 34.89%, 12.77% and 9.55%": higher or lower as compared to control?
LN229-234: may be moved to the Materials and Methods section
LN251-259: may be moved to the Materials and Methods section
LN324-334: may be moved to the Materials and Methods section
LN504: check the statement
LN567: check the statement
Author Response
Dear professor,
On behalf of co-authors, we are very grateful to you for giving us an opportunity to revise our manuscript. We appreciate you very much for your positive and constructive comments and suggestions on our manuscript. Your feedback has been extremely helpful in revising and improving our paper, as well as guiding our research.
We have carefully considered your suggestions and tried our best to revise our manuscript according to the comments. The revised portions are highlighted in red within the paper. Thanks again to the hard work of reviewer!
Response to the comments
Comment No. 1: LN39: Selenium may be termed a 'beneficial element', instead of 'micronutrient'.
Response: Thank you very much for your suggestion. We have replaced the “beneficial element” with “micronutrient” in section 1 (p2 LN43).
Comment No. 2: LN62-63: rephrase the sentence.
Response: Thank you very much for your suggestion. We have modified the sentence (The pak choi (Brassica chinensis var. pekinensis. cv. ‘Suzhouqing’) is rich in fiber, vitamins and mineral elements) in section 1 (p1 LN38-39).
Comment No. 3: LN83: incomplete.
Response: Thank you very much for your suggestion. We have added some specific details as shown in page 2 LN68-70: Guo et al. found structural genes, phosphate transporters and sulfate transporters that may be involved in selenium metabolism when analyzing Pueraria lobata (Willd.) Ohwi response to selenium stimulation at molecular level.
Comment No. 4: LN88-91: rephrase the sentences.
Response: Thank you very much for your suggestion. We have modified the introduction to make our objectives more clear, as shown in page 2 LN68-70: Therefore, we used transcriptome sequencing analysis to identify differentially expressed genes and analyze the enriched metabolic pathways associated with these genes. The physiological and biochemical mechanisms and molecular regulatory mechanisms of leaf surface application of selenium to low temperature stress were finally revealed, which provided a theoretical basis for further enhancing yield and quality and increasing selenium content.
Comment No. 5: LN 94 and 98: check the mentioned date.
Response: We are sorry that we have not expressed the experimental conditions correctly and clearly, and thank you very much for your suggestion. We have rewritten the experimental design, as shown in page 2 LN 86-94 : On December 19th, the seedling plants were moved into an artificial climate chamber under low temperature stress conditions with a light intensity of 2000 lx, photoperiod of 12 h/12 h, temperature of 2 ℃ and relative humidity of 75%. on December 9th, 16th (prior to being transferred into the artificial climate chamber) and on December 27th (after being transferred into the artificial climate chamber), leaves were sprayed with 20 mL of water (LCK), 20 mL nano-selenium at concentrations of 5 mg L-1 (LN5),10 mg L-1 (LN10) and 20 mg L-1 (LN20), as well as 20 mL organic selenium at concentrations of 5 mg L-1 (LO5),10 mg L-1 (LO10) and 20 mg L-1 (LO20).
Comment No. 6: LN146 and elsewhere: how do the authors justify the increase in SOD activity by 341.16%? When the percent increase is more than 100, better to express it in fold-change units.
Response: Thank you very much for your suggestions. We have used fold-change unites when the percent increase was more than 100 as shown in section 3.2 (p5 LN 195-196, 204-205) and 3.4 (p6 LN243-244).
Comment No. 7: LN178-179: "In comparison to control, GSH content sprayed with organic selenium was 34.89%, 12.77% and 9.55%": higher or lower as compared to control?
Response: Thank you very much for your suggestions. We have revised the sentence as “In comparison with the control group, the GSH content sprayed with organic selenium increased 34.89%, 12.77%, and 9.55%, respectively.” (p5 LN227-229).
Comment No. 8: LN229-234: may be moved to the Materials and Methods section. LN251-259: may be moved to the Materials and Methods section. LN324-334: may be moved to the Materials and Methods section.
Response: Thank you very much for your suggestions. We have moved these parts into the Materials and Methods section, which are section 2.4, 2.5 and 2.6 (p3 LN121-160).
Comment No. 9: LN504: check the statement.
Response: Thank you very much for your suggestions. Since this content was already presented in the results section, it was removed from the discussion section.
Comment No. 10: LN567: check the statement.
Response: Thank you very much for your suggestions. We have revised the sentence as “It has been hypothesized that transporters related to plant cysteine methionine may be involved in some processes of organic selenium absorption” (p20 LN606-608).
Author Response File: Author Response.docx
