Genome-Wide Identification of miRNAs and Its Downstream Transcriptional Regulatory Network during Seed Maturation in Tilia tuan

Round 1

Reviewer 1 Report

The topic is interesting.  I see two main concerns: miRNA precursor prediction and transcript level determination are poorly documented or not even mentioned in the material and methods section, making a rigorous evaluation impossible. Figures - especially supplemental figures - need more precise legends, other ways they are appropriate. Tables are some minor mistakes. References are extensive, some of the applied software are not referenced, this should be corrected. For statistical analysis the authors used built-in analyses of the applied software. However, sample constitution and replications are not presented clearly. In addition to the mains concerns formulated, data analysis is documented generally relatively poorly, in most cases the applied software is given, but not the settings/algorithms.

Lines 50-51: miRNAs were first discovered in plants. AFAIK this statement is false. The first miRNA (lin-4) was discovered in Caenorhabditis elegans. "

Lines 64-65: four species, three references - which one refers to which one?"

Lines 66-67: Which species?"

Lines 70-71: How is MYB89 connected to miRNAs?"

2.1. Plant materials：Please provide exact developmental stages of the sampling times based on a standardized phenological scale - if available - or definition of the developmental stages. Sample management is unclear, please elaborate."

Lines 93-94: - How many seeds formed one sequencing sample? Did You use whole seed extracts?"

Sequence data analysis: Which software was used for sequence filtering? Or was it done "by hand"? Please provide this information."

Line 110: Please define the reference sequence.

Line 173: This section implies that additionally to small RNA sequences, you have RNASeq/Microarray data as well. However, this isn't mentioned in the materials and methods section."

Lines 332-337：It is still not clear where the RNA expression data is coming from"

Lines 398-400：Which way did mir160 change. Is this connected to ABA2?"

Lines 448-462：In this part, fatty acid synthesis (which was discussed already two pages above) and lignin biosynthesis are mixed. Please clarify. Also, lignin biosynthesis aspects are discussed one page later."

Table S7: Why is TPM information missing? It is listed for known miRNAs in TableS5"

Author Response

“Reviewers' comments:

1. Reviewer #1: " Lines 50-51: miRNAs were first discovered in plants. AFAIK this statement is false. The first miRNA (lin-4) was discovered in Caenorhabditis elegans. "

Our response: We thank Reviewer 1 for this constructive comment. This sentence has been revised in current manuscript.  

 

For instance: “In 1993, a small RNA with negative regulation was first identified while studying the embryonic development of Caenorhabditis elegans [8]. In 2002, miRNAs were first discovered in plants [9].”

 

2. Reviewer #1: " Lines 64-67: four species, three references - which one refers to which one?"

Our response: We thank Reviewer 1 for this constructive comment. This sentence has been added two references and revised in current manuscript.

 

For instance: “The involvement of miRNAs in post-transcriptional regulation of seed and fruit development has been documented in apricot [20], rice [21], soybean [22], and Brassica napus [23].”

 

3. Reviewer #1: " Lines 66-67: Which species?"

Our response: We thank Reviewer 1 for this constructive comment. It is in Arabidopsis specie.

 

For instance: “miR160 negatively regulates auxin response factors involved in Arabidopsis seed development [27] and floral organs of Arabidopsis [28]”

 

4. Reviewer #1: " Lines 70-71: How is MYB89 connected to miRNAs?"

Our response: We thank Reviewer 1 for this constructive comment. There is no regulatory relationship between MYB89 and miRNAs in this reference article. This sentence is only to illustrate the regulatory role of transcription factors.

 

For instance: “MYB89 functions as a negative regulator of seed oil accumulation during maturation in Arabidopsis seeds [30]”

 

5. Reviewer #1: " 2.1. plant materials: Please provide exact developmental stages of the sampling times based on a standardized phenological scale - if available - or definition of the developmental stages. Sample management is unclear, please elaborate."

Our response: We thank Reviewer 1 for this constructive comment. I am sorry that it's not available. This sentence has been revised in current manuscript.

 

For instance: “The T. tuan seeds were collected from an individual T. tuan with good growth and development, at Beijing Forestry University. Five seed samples (Jun. 1, June 1, 2019; Jul. 1, July 1, 2019; Aug. 2, August 2, 2019; Sept. 2, September 2, 2019; and Oct. 2, October 2, 2019) were obtained in T. tuan at different seed maturation stages (Figure 1A). Three independent biological replicates of thirty seeds at each maturation stage were collected and immediately frozen in liquid nitrogen, and stored at -80°C for further analysis.”

 

6. Reviewer #1: " Lines 93-94: - How many seeds formed one sequencing sample? Did You use whole seed extracts?"

Our response: We thank Reviewer 1 for this constructive comment. Thirty seeds formed one sequencing sample and we used whole seed extracts.

 

7. Reviewer #1: " Sequence data analysis: Which software was used for sequence filtering? Or was it done "by hand"? Please provide this information."

Our response: We thank Reviewer 1 for this constructive comment. Raw data (raw reads) of fastq format were firstly processed through custom perl and python scripts.

This sentence has been revised in current manuscript.

 

8. Reviewer #1: " Line 110: Please define the reference sequence"

Our response: We thank Reviewer 1 for this constructive comment. The resulting full-length transcripts used as a reference sequence used to identify and quantify genes.

 

9. Reviewer #1: " Line 173: This section implies that additionally to small RNA sequences, you have RNASeq/Microarray data as well. However, this isn't mentioned in the materials and methods section."

Our response: We thank Reviewer 1 for this constructive comment. We have RNASeq/Microarray data, which has been mentioned in the materials and methods section. At the same time, we have been deposited at the National Center for Biotechnology Information Sequence Read Archive (NCBI, SRA, http://www.ncbi.nlm.gov/sra/) under accession number PRJNA861431 and PRJNA861958.

 

10. Reviewer #1: " Lines 332-337：It is still not clear where the RNA expression data is coming from"

Our response: We thank Reviewer 1 for this constructive comment. The RNA expression data is coming from RNA-Seq data. We have been deposited at the National Center for Biotechnology Information Sequence Read Archive (NCBI, SRA, http://www.ncbi.nlm.gov/sra/) under accession number PRJNA861958.

 

11. Reviewer #1: " Lines 398-400：Which way did mir160 change. Is this connected to ABA2?"

Our response: We thank Reviewer 1 for this constructive comment. This sentence has been revised in current manuscript.

 

For instance: “ABA2 is not expressed from Jun. 1 to Aug. 2 stage, however it is significantly upregulated at Sept. 2 and Oct. 2 stage. The expression levels of miR160 altered dynamically from Jun. 1 to Oct. 2 stage, with the highest level occurring at Oct. 2 stage.”

 

12. Reviewer #1: " Lines 448-462：In this part, fatty acid synthesis (which was discussed already two pages above) and lignin biosynthesis are mixed. Please clarify. Also, lignin biosynthesis aspects are discussed one page later."

Our response: We thank Reviewer 1 for this constructive comment. In this part, fatty acid biosynthesis aspects are discussed. This word has been replaced “lignin” to “lipids” in this sentence.

 

For instance: “Thus, it is speculated that this miRNA negatively regulates lipids synthesis.”

 

13. Reviewer #1: "Table S7：Why is TPM information missing? It is listed for known miRNAs in TableS5"

Our response: We thank Reviewer 1 for this constructive comment. We have added TPM information in TableS7.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments to the authors:

Authors analyzed miRNA at the genome-wide level and identified its downstream transcriptional regulatory network during seed maturation in Tilia tuan. This is an interesting study, which provide new cues to further uncover the mechanism of miRNA during seed maturation in Tilia tuan and other similar species can also be used for reference. The MS should be published after revised.

 

All my questions about this article are as follows：

1.       Neither the authors nor me are native speaker. However, there are numerous other smaller issues, but it would be easier to address these once this paper is in better shape.

2.       Authors should add more recent references in introduction and discussion section, about 3-5 years.

3.       Line153, The author is requested to carefully review the spelling of the words in the manuscript for accuracy, such as “count s”.

4.       I am not a forest scientist, so I don't know whether Tilia tuan can be genetically transformed, if Tilia tua can be genetically transformed like Populus L., the research value of this result will be even greater.

Comments for author File: Comments.docx

Author Response

14. Reviewer #2: "Neither the authors nor me are native speaker. However, there are numerous other smaller issues, but it would be easier to address these once this paper is in better shape."

Our response: We thank Reviewer 2 for this constructive comment. There are numerous other smaller issues has been revised in current manuscript.

 

For instance: “MYB89 functions as a negative regulator of seed oil accumulation during maturation in Arabidopsis seeds [30].” “Jun. 1, Jul. 1, Aug. 2, Sept. 2 and Oct. 2 represent T. tuan seed collected at June time point, July time point, August time point, September time point, and October time point 2019, respectively.” “Genes were assigned into three main categories: biological processes, cellular components and molecular functions.” “(A) Represent targets functions of miRNA involved in fatty acid biosynthesis, Heat maps of gene expression levels (FPKM) at five different seed maturation stages. (B) Heat map of expressed miRNAs involved in fatty acid biosynthesis. (C) Represent the view of miRNA–mRNA network involved in fatty acid. The red color represents miRNA and the blue color represents targets.”

 

15. Reviewer #2: "Authors should add more recent references in introduction and discussion section, about 3-5 years."

Our response: We thank Reviewer 2 for this constructive comment. We have been added recent references and revised in current manuscript.

 

16. Reviewer #2: "Line153, The author is requested to carefully review the spelling of the words in the manuscript for accuracy, such as “count s”."

Our response: We thank Reviewer 2 for this constructive comment. This sentence has been revised in current manuscript.  

 

For instance: “Furthermore, the miRNA counts as well as base bias on the first position of identified miRNA with certain length and on each position of all identified miRNA were obtained respectively.”

 

17. Reviewer #2: "I am not a forest scientist, so I don't know whether Tilia tuan can be genetically transformed, if Tilia tua can be genetically transformed like Populus L., the research value of this result will be even greater."

Our response: We thank Reviewer 2 for this constructive comment. Tilia tuan can be genetically transformed like poplar, which is the direction of our further research in the future. At present, we are studying the genetic transformation in Tilia tuan and believe that the genetic transformation system will become mature one day in the future.