Analysis of Relative Expression of Key Enzyme Genes and Enzyme Activity in Nitrogen Metabolic Pathway of Two Genotypes of Potato (Solanum tuberosum L.) under Different Nitrogen Supply Levels
Round 1
Reviewer 1 Report
The manuscript is quite nice to read although some parts could be shorter and some important information is missing.
1) Abstract
- Although it may be well known - explain the enzyme abbreaviations first (NR, NiR, GS, GOGAT, GDH). On the other hand, nitrogen could be abbreviated to N. In the text body, N is used for nitrogen inconsistently.
2) Material and Methods
- Which genes were used as reference? It is not clear from the table 1.
- The enzyme activity measurement must be better explained. At least which substrates, if it was measured spectrofotometrically etc. I don't think that 1 kit is used for all these enzymes. The name of the kit is also missing...
- A chapter about statistical evaluation must be added.
3) Results
- All figures must have better legends. All abbreaviations must be explained here as well (S, B, T, M). Also, the quality must be better (bigger pictures) because +/- can be barely seen. Also, statistics (meaning of letters above columns) must be explained. Figure names should clearly name the enzyme as well (not only the gene).
- Enzyme activity figures - what is "g"? Is it fresh weight or what? It should be explained in the legend. The same goes for micromol. Micromol of what? Statistics is missing for these graphs. Also, better explain "under different nitrogen supply levels" (N+, N, N-).
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Manuscript presented to evaluation concerns analysis of key enzyme genes and activity in nitrogen metabolism pathway. The expression and activity were analyzed in leaves and roots of two potato genotypes differing in nitrogen efficiency. The studies were conducted in plants grown in three different nitrogen supply and in four different plant development stages. For this reason there are many results presented in the manuscript. However, the results are not very knew.
Abstract: The first three sentences should be rewrite in present simple, such as ‘Nitrogen plays an important role ….’ and so on, because nitrogen still do it and do it all the time. The sentence in Line 27-31 should be removed to the line18, before the results were described.
Key words should be changed. Relative expression quantity and enzyme activity analysis are not good key words in my opinion. They are too general.
Introduction: the sentence in L67-68 (In addition, more than 90% of nitrate absorbed by crop roots is assimilated into roots and stems by nitrogen ….) is unclear.
The abbreviation ‘KEGG’ must be explained on the first use in the introduction (Line75).
The authors listed all the reactions of the nitrogen metabolism pathway. However, not all of these occur in plants. This should be clarified.
The last paragraph contains too much methodological information, which can be moved to Materials and methods as explanation of experiments treatments and analyses performed. Instead, the authors try to provide hypothesis what was expected to be found.
Results:
References to the previous research mention in paragraph 3.1 should be added.
The results of the relative gene expression presented in the figures1-14 are not clear in some cases. There are so many of them that it is difficult to present them in a clear way and to analyze them. The order in which the results are presented in the bars on the figures should be the same. This should make it easier to analyze the results in the figures by the readers.
The scale on the figures showing enzyme activity should be the same for the results of both potato genotypes studied. Then you will see the difference in activity levels between these two plants.
There is a mistake in the sentence L421-422. Nitrite reductase (NiR) can reduce nitrite (NO2-) produced by nitrate reductase, to form NH4+. Nitrate (NO3-) absorbed from the soil is firstly reduced by nitrate reductase (NR). I suggest, to enter a sentence explaining what the role of each studied enzyme is in the nitrogen metabolism pathway.
Discussion is somewhat chaotic and disorganized, possibly due to the sheer amount of results and should be rewrite. Moreover, the authors performed analyzes of genes expression and enzyme activity at 4 different development stages of the potato and described them extensively in the results. However, in my opinion, they do not refer to it at all in the discussion. They only consider the general differences between the different levels of nitrogen nutrition.
Line 550-551: dry matter was not analyzed in this study so probably a reference is needed.
Conclusion is not very knew. It has long been already known that the enzymes involved in the assimilation of inorganic nitrogen in plants are nitrogen induced and their activity increases with the availability of mineral nitrogen in the soil.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you. The manuscript was significantly improved.
Reviewer 2 Report
All of the reviewer’s comments mentioned in the previous revision were included in the manuscript. In my opinion, it is now significantly improved. Thank you for all explanations. I accept them.
