Effect of Low R:FR Ratio on Nitrogen Assimilation and NRT Gene Expression in Pakchoi under Excessive Nitrate Stress

Round 1

Reviewer 1 Report

Nitrogen metabolism of plants is relevant, and the study contain true information about it. I made comments to try to make the manuscript easier to be read. I apologize in the cases I may have misunderstood the authors.

Line 32 In a greenhouse it seems necessary to have water input.  Is this is rarely sufficient? Please, give more detail.  The cultivated in greenhouse is for human nutrition only?

Lines 40-41 something seems to be missed

53 NiR is nitrate reductase? It was as NR before.

59 the paragraph may begin with ‘Nitrate transporter gene family’ ?

63 NTRs constituents of  biological cell plasma membrane ?

64 I did not understand ‘systematic’

66 genes (plural)? After the point, use capital letter

67 I did not understand this line. The effect of variations in the ratio on the gene family expression? Please, improve.

68 to 72 English writing style must be improved. I expect in this manuscript you won’t base the conclusions on a single experiment, anyway, this will be described later. So I suggest the authors should not begin the paragraph with ‘in this experiment’. And the way you describe it give me a first impressions that controls are missed. I know you did not miss them, I read, it is okay. I suggest you try to write again, because this paragraph is important to the reader.

75 to 84 I would expect to be informed about the size of the tray and substrate volume. Please justify why you have chosen this single nitrate source.

85 Describe better the light wavelengths ratios. When is it similar to sun ratios, is there an amplified one, which of them?

87 what is normal? Other nutrients may have an influence.

92 How many samples per treatment?

93 How nice you made three experiments. How many seedlings per treatment?

95 `Please , briefly describe plant growth measures, and also the others.

104 To which species have been the primers designed?

113 I think ANOVA and Duncan are different steps, I am not used to describe both as a single step analysis.

Table 1: Are roots included? Make treatments easier to read. Factorial analysis was not used? Use one line to each of the treatments, because the legend is essential and difficult to follow.

132 significantly according to Duncan test, not using a factorial design?

134 was? The chlorophyl content increased at?

199 When you say salt, for me is a kind of general term, and the first salt I would have in mind with this term is NaCl, or others, like aluminum salts. I leave in a reality where NO3 is required, not in excess. Which salts are these that the authors refer here?

212 which plant species?

221 in these three assays reported in this paper? The authors  tested very young plants. What about the other reports?

Author Response

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Reviewer 2 Report

The presented work reports on the role of low R:FR ratio on nitrogen metabolism of Pakchoi under excessive nitrate stress and different physiological parameters such as growth index, chlorophyll content and key enzymes of nitrogen metabolism, nitrate and glutamic acid content and NRT gene expression level of  pakchoi leaves were examined. The results indicated that low ratio of R:FR could alleviate the reduction of growth and chlorophyll content in pakchoi under stress of high-level nitrogen (H80 and 16 H160). Activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamine synthetase (GOGAT) decreased under H80 and H160, except of NR when under the H80 treatment.

Authors need to focus on the following sections:

-          Introduction is not reflecting the problem addressed, its worth elaborating

-          Results graph needs improvements; shading is not clear in the bar graphs.

-          Discussion: Line # 211: Our previous study [2] pointed that the low ratio of R:FR could promote the activity, reference number is incorrect.

Overall, the manuscript needs a through revision to address typographical and spelling mistakes.

Author Response

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Reviewer 3 Report

The manuscript is well planned and presented. However a few suggestions/corrections need to be incorporated and are as follows:

Line and bar diagrams presented in the manuscript are not given any title. Also, these are not clearly visible/legible. This need to be corrected.

line 29: Brassica campestris should be made italics

Line 75: proceeded should be converted to 'conducted'

line 76: change 'from' to 'during'

line 81: Correct ((

line 132: change 'significantly' to 'a significant'

line 135: change 'then' to 'and then' 

The conclusion section is too generalized and should be modified to more oriented writing.

Overall, the language of the manuscript needs to be carefully checked for grammatical mistakes (a few are marked).

Comments for author File: Comments.pdf

Author Response

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Round 2

Reviewer 1 Report

I intend to give just some small suggestions

Line45 (suggestion): instead of saying ‘is not enough’, you could indicate which result was a need accomplished by the manuscript

Is reference 14 a thesis? Just a suggestion, to ‘complete the reference if possible.

Line 82:  the three assays?

Legend Figure 1 and methods lines 97 to 106. I suggest to make the experimental design easier to read. At methods,

(1)  CK: normal nutrition, (is this Hoagland solution ?) R:FR = 4.2;

(if R:FR ratio is approximately 1.15 under natural light sources, this control is not similar to natural environment. This must be clear from the beginning at results, since they results are presented before methods in the text. I apologize if I am misunderstanding the authors.

You used two R:FR ratios x three salt concentrations, where 80 and 160 indicate the salt doses, and H and L indicate the two R:FL ratios. I do not see it at the first glance when I read results, and I delay to read it at methods. It is okay if you did not analyze it as a factorial design.

Author Response

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