Nitrogen Absorption Pattern Detection and Expression Analysis of Nitrate Transporters in Flowering Chinese Cabbage

Round 1

Reviewer 1 Report

The article entitled "Nitrogen Absorption Pattern Detection and Expression 2 Analysis of Nitrate Transporters in Flowering Chinese 3 Cabbage" is a comprehensive study related to nitrate transporter in Chinese cabbage. However, it needs to address major comments and English language and grammatical correction.

Major comments:

Q1: Authors used "The nutrient solution formula was as follows: 4.0 mmol/L NaNO3, 2.0 mmol/L KH2PO4, 130 2.0 mmol/L KCl, 2.0 mmol/L MgSO4, 0.5 mmol/L CaCl2, 0.1 mmol/L Fe-EDTA, 50 μM 131 H3BO3, 12 μM MnSO4, 1 μM ZnC12, 1 μM CuSO4, 0.2 μM Na2MoO4, 30mg/L ampicillin." why they used ampicillin here ?
Q2: For treatment authors used nitrogen starvation and then with different concentrations of NaNO3 (2, 3, 4, and 8 mM) and NH4Cl (1, 3, 4, and 8 mM). why did they choose this 2-8mM concentration? put any reference.

Q3: Figure 1 b should be reconstructed with more NRT genes in various plants which are reported. 

Q4: Authors stated that "The specific primers of three BcNRT genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) are designed based on the sequence of Arabidopsis (Table S1)" at line 146. Rewrite it again not clear.
Mention here the NCBI accession no here and Table S1.

Minors comments
Line 57: gene name should be italics as written in line 50 
Line 60, 63 Write Arabidopsis in italics throughout the manuscript.
Line 106 -114 put the appropriate references
Line 116 mentioned the scientific name also in bracket "Brassica campestris" along with the English name at line 106.
line 148 Write "The full length or CDS seq of BcNRT genes were amplified using PCR with specific primers."
line 195 Fig1c should be re draw with increased font size. not visible.
Line 198 Zea mays should be italics.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The authors isolated three NRT genes in flowering Chinese cabbage based on Arabidopsis NRT sequence and analyzed their expression patterns in the different tissue and developmental stage. In addition, they analyzed the expression patterns and N uptake rate after applying the different N concentrations and sources. Overall, the experiments were designed and performed well and the manuscript was written well. However, I have several questions and the authors have to clarify them to publish the manuscript.

1. In 2.3 (line 167), what is the CK? Can you explain it?
2. The authors described that Actin and GAPDH were used as an internal control for qRT-PCR in 2.5. I wonder whether both genes were used at the same time or separately for each experiment. If you used them separately, you have to describe which gene was used as an internal control in the legend of each figure.
3. The authors showed the sequence alignment only for NRT1.1 in Figure 1C. Alignment data for the other two NRTs have to be included.
4. There were discrepancies between ORF length and amino acid length (Line 205-208)
5. For N uptake and absorption analysis, which parts of the plants were used? Whole plants or roots? The authors have to describe it in each figure legend.
6. In Figures 3 and 4, describe which parts of the plants were used for NRT expression analysis.

Minor points

1. Across the manuscript, there are many errors in superscripts (ex. NH4+, NO3-). Check the superscripts
2. Check the sentence in Lines 351-352.
3. In Lines 481-483, there is no (2).

Author Response

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Reviewer 3 Report

In the present manuscript the authors investigate the relation between the expression of NRT genes in Chineses cabbage and nitrogen uptake properties. In my opinion the general interest in the presented data are low, and also specific interest will be limited. Additionally, there are several points which have to be corrected before consideration of publication.

Points to be corrected:

• Fig. 1C is far too small to be read. I would suggest to shift it to the supplement together with Fig. 1A, as both do not contribute directly to the understanding of the main text. Additionally, I would suggest to enlarge Fig. 1B.
• The error bars in Figs. 2-4 are not explained. Please correct!
• The scales of the y axis of Figs. 3C, 3D, and 4C are different between the NRTs. Please use one scale within one line.
• In these figures regression lines have been drawn between the data points, but the time axis is not in scale, which needs to be corrected.
• The term "extremely significantly" in line 332 is inapropriate.

In addition to these points I have several questions and remarks about the text:

• line 203: What does "target bend" mean?
• lines 209-217: Are there more NRTs encoded in the genome of B. campestris? Then they should be included in the phylogenetic analysis as well as additional NRTs from the other plants. Using all available sequence information enables correct ordering of the proteins of interest.
• The section in lines 230-246 is too long and should be shortened.
• I must admit that I missed the meaning of the section in lines 259-264.
• What kind of "overall upward trend" is in the lines 265-267?
• What are your arguments that "physical and chemical properties" are similar between NRTs of Arabidopsis and Chinese cabbage (lines 347-348)?
• In lines 481-483 you wanted to raise three questions, but no. 2 is missing.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Three little comments:

1. In line 133 you call ampicillin a fungicide, but it´s an antibiotic.
2. In line 215 there is a typo which turns NRT2.1 into 1.2.
3. There are still lots of other typos and the grammar also needs some polishing.