Comments on abstract, title, references

Is the aim clear? The authors, clearly express the goal of the study in the 7th sentence of the abstract. "The aim of this study was to determine how nitrogen repletion affected the transcriptional response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour after nitrogen addition."

● Is it clear what the study found and how they did it? “We found almost 4800 genes induced or repressed, sometimes within minutes after nutrient changes. Some of these responses to nitrogen depended on the TOR pathway, which controls positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, and genes related to the retrograde response (RTG) specific to the tricarboxylic acid (TCA) cycle and nitrogen catabolite repression (NCR). Some unexpected transcriptional responses concerned all the glycolytic genes, carbohydrate metabolism and TCA cycle-related genes that were down-regulated, as well as genes from the lipid metabolism.” ● Is the title informative and relevant? Concerning to the tittle, it’s perfectly resuming what they found and it completely informs about the statement the authors want to show. The title could be relevent not only for the researcher of the field but for the wineries as well. ● Are the references: ● Relevant? All the references they present are very specific and important for this study as most of them are about the nitrogen assimilation during fermentation, glucose and lipid metabolism, the wine yeast strain used in this study and the gene expression. ● Recent? The most recent references date in 2017, so it's enough recent but I’d like to have more recent studies like doctoral thesis or book chapters at least. ● Referenced correctly? the format is correct and clear in the references list. ● Are appropriate key studies included? All the current similar studies based on Nitrogen starvation are included.

Comments on introduction/background

● Is it clear what is already known about this topic? Yes, it is. The authors make a good resume about what it’s already known about the Nitrogen limitation and it’s consequences at the metabolic level. ● Is the research question clearly outlined? The authors say “Our goal was to detect new phenomena appearing quickly after nitrogen addition” So it’s clear that they want to analyze what happens just after the nitrogen relief.

● Is the research question justified given what is already known about the topic? The authors present the gap in knowledge that exist: “Although this nitrogen addition is currently practiced using diammonium phosphate to reduce the risk of stuck fermentation in white and rose wines, the molecular mechanisms triggered by nitrogen replenishment are still poorly understood”.

After they say how they can fill this gap: “The present work complements previous investigations on laboratory [9] or enological [10] yeast strains with the novelty of transcriptome analysis every 15 min during the first hour following relief of nitrogen starvation in a medium mimicking grape must composition with limiting nitrogen concentration.”

Comments on methodology

Comments on methodology

● Is the process of subject selection clear? The authors say that they use the EC1118 yeast wine strain. This is a very known strain in the field of wine yeast so they exclude any other comments or justification in the strain methodology section as they took for granted that the reader is going to understand why they choose this strain. ● Are the variables defined and measured appropriately? The authors need to analyze genome expression for some time points after Nitrogen starvation (t=0) and nitrogen addition (other time samples). So, the variables are very clear: time after nitrogen addition and fold change in expression. ● Are the study methods valid and reliable? The methods employed here are the genome expression analysis by ChIP array followed b a bioinformatic analysis with an R package (limme). This methodology is well known and very commonly used in this field and other to carry on this analysis. They are in general validated for the science community and the can be repeated in other labs as they give all the conditions information. ● Is there enough detail in order to replicate the study? Any other research can follow the steps shown in the paper to replicate the experiments.

Comments on data and results

● Is the data presented in an appropriate way? The results of this paper are about the upregulated and the downregulated genes after the DAP addition during 1h. The authors present the data in an appropriate way as they first mention the number of genes found for each group and after they group the genes in each category depending on how and when the expression is changing (for instance, if the expression only change in the first time point and after is linear). They after mention the gene ontology categories that they found in each group and the genes inside them. ● Tables and figures relevant and clearly presented? For figures 2 and 3, although they clearly explain how is each one of the group’s behavior concerning to gene expression, I’d like to see a little title above each graph to know what it’s referred to or in the figure footnotes. For figure 4, any other footnotes or explanations in the results text are necessary that justify why they choose these glycolytic genes and no others. Why the authors chose these 16 genes? Are the only ones presenting changes? Are the relevant?

● Appropriate units, rounding, and number of decimals? Units and decimals in numbers either in figures and text are properly written.

● Titles, columns, and rows labelled correctly and clearly? All of this is properly written.

● Categories grouped appropriately? Categories concerning up-regulated and down-regulated genes and subgroups inside them are perfectly clear about the criteria chosen for group them.

● Does the text in the results add to the data or is it repetitive? the text explaining the results is adequate, it’s not redundant in any case. ● Are you clear about what is a statistically significant result? Although the preprint is lacking this information for the short of the format, the authors are going to add all the statistical and numerical information in supplemental data and in public repositories. ● Are you clear about what is a practically meaningful result? Meaningful results are clear as the authors pinpoint several times what is important in the results they found.

Comments on discussion and conclusions

● Are the results discussed from multiple angles and placed into The authors properly blended the results with the discussion section as they can compare in a better way the older and the new results. The results in change in gene expression on metabolic genes is well discussed and well joined to the cellular changes that are occurring at each time point.

● Do the conclusions answer the aims of the study? Yes, it does. The conclusions clearly show some new results that were not found in previous works by the study make here. ● Are the conclusions supported by references or results? The authors employ the references to compare what they have obtained with previous results so they share some results with others, as in the case of CO2 production after nitrogen addition to the media or the global response in up-regulated genes and in the TOR pathway. They also found some similarities with other works in the downregulated category. They also employ the references to support they experimental design. The authors also compare the number of genes obtained in three different studies, so they attribute the better performing on their methodology that reason of obtaining a higher number of genes that change their expression.

● Are the limitations of the study fatal or are they It’s a lacking auto-evaluation of the study limitation which is desirable in every study.

1)Overall statement or summary of the article and its findings in your own words This works provides new information about fold change transcription in metabolic genes, overall, in glycolytic genes after nitrogen addition in a nitrogen depleted synthetic must media. In my opinion, the paper is well written in general and the new information concerning to fermentative process is always welcoming for the wine industry. From the scientific point of view, it’s is always desirable to go deep in the biological repercussion and the limitations of the study.

2)Overall strengths of the article and what impact it might have in your field. The authors show how the improvement on methodology directly impacts on the results obtained. I think that this is the highest strength of the paper.

3)Specific comments on weaknesses of the article and what could be done to improve it. Major points in the article which needs clarification, refinement, reanalysis, rewrites and/or additional information and suggestions for what could be done to improve the article.

1. They should add the limitations of this research work in the discussion section and comment some other research that they could possibly include in the paper to make it more interesting and complete.
2. There is a lacking on experimental data, so they can add some variables or play with the expression of some genes to see if they obtain interesting changes.

Minor points like figures/tables not being mentioned in the text, a missing reference, typos, and other inconsistencies. 1. They should add more information in some figures footnotes as it was mentioned before. 2. 3.