Exogenous DCPTA Increases the Tolerance of Maize Seedlings to PEG-Simulated Drought by Regulating Nitrogen Metabolism-Related Enzymes

Round 1

Reviewer 1 Report

In their manuscript "Exogenous DCPTA increases the tolerance of maize seedlings to PEG-simulated drought by regulating nitrogen metabolism-related enzymes" Xie et al. aim at unravelling the molecular basis of 2-(3,4-Dichlorophenoxy)triethylamine (DCPTA) mediated drought tolerance. The group had previously shown that DCPTA application could increase drought tolerance in Maize by improving photosynthetic capability and regulating the antioxidant system and nitrogen metabolism. They employ a system in which drought is simulated by application of Polyethyleneglycol (PEG-6000) to medium in hydroponic cultures. Plants submitted to this simulated drought stress were compared to control plants (medium only) and both treatment types were carried out with and without DCPTA. As expected from previous results of the group, plants simultaneously treated with PEG and DCPTA showed reduced drought phenotypes and less growth reduction compared to PEG only treated plants. To identify genes putatively affected by DCPTA treatment, an RNA-seq analysis was performed, which revealed several genes coding involved in metabolic processes, including genes coding for enzymes involved in nitrogen metabolism as differentially expressed. Those candidate enzymes showed reduced protein abundance and activity upon PEG treatment, which was partially reverted by DCPTA treatment. The same was observed for nitrate and nitrite levels, soluble protein levels and amino acids per dry weight. Thus, the authors could identify several molecular effects of DCPTA likely contributing to drought tolerance.

Although the data is potentially interesting to the Maize community and also to drought stress researchers in general, some issues need to be clarified before this manuscript could be published:

- Experimental design: please describe the experimental design in more detail. Mention if pot positions were randomised and most importantly how many plants were used and if biological replicates were performed. This information is needed for every experiment, including the RNAseq.

- Table 3: A sample number of 5 seems very small

- Fig 3 only shows absolute numbers, no comparison to the background population of all genes, but the text claims there is an enrichment. This is not shown in the figure.

- Table 4: is there a way to create a graphical representation to highlight the most important results? The large tables are challenging to read.

- Fig 4: five technical or biological replicates? Which test was used? Should be stated for all figures.

Minor points:

- Abstract line 11: it is hard to understand which DEGs are meant here as the explanations follows at the end of the sentence.

- Introduction lines 70/71: I do not really see the difference between points 2 and 3

- Results line 105: separate the numbers by ; instead of , to avoid confusion

- Fig 5: please refer to protein abundance rather than expression

- Results line 278/279 please state in which comparison the upregulation was observed

- Adding an introductory sentence stating what was done and why at the start of each paragraph would make the manuscript easier to follow.

- Discussion line 378: RNAseq does not measure transcription but mRNA levels

- Discussion lines 424-430: Please explain the purpose of this paragraph. It sounds like a description of the method performed in this work but does this mean the previously performed method is inadequate? What is meant by existing method? The one used in this work?

Author Response

Comment: Experimental design: please describe the experimental design in more detail. Mention if pot positions were randomised and most importantly how many plants were used and if biological replicates were performed. This information is needed for every experiment, including the RNAseq.

Reply: Materials and methods section, line 93, 108-118, page 2-3 in the revised manuscript, we have mentioned container positions, the number of plants were used;

Comment: Table 3: A sample number of 5 seems very small.

Reply: Results section, Table 3, line 230-240, page 5-6 in the revised manuscript: In our previous statistics, the mean values of 10 plants from one container were considered one replication, as a result, the sample number was 5. We have realized that the approach is not accurate, therefore, the value of 1 plant was considered one replication in our present statistics, the sample number was 50;

Comment: Fig 3 only shows absolute numbers, no comparison to the background population of all genes, but the text claims there is an enrichment. This is not shown in the figure.
Reply: Results section, Fig 3, line 277-280, page 7 in the revised manuscript, we have shown the comparison of DEGs population to the background population of all genes in the figure; Comment: Table 4: is there a way to create a graphical representation to highlight the most important results? The large tables are challenging to read.

Reply: Results section, Table 4, line 289-295, page 7-11 in the revised manuscript, we have realized that the large tables are challenging to read, and highlighted the most important results with bold type;

Comment: Fig 4: five technical or biological replicates? Which test was used? Should be stated for all figures.

Reply: Results section, Fig 4-9 in the revised manuscript, we have stated five biological replicates and LSD test for these figures;

Comment: Abstract line 11: it is hard to understand which DEGs are meant here as the explanations follows at the end of the sentence.

Reply: Abstract section, line 14-18, page 1 in the revised manuscript, we have amended the explanations to avoid the hard understand;

Comment: Introduction lines 70/71: I do not really see the difference between points 2 and 3.

Reply: Introduction section, line 85, page 2 in the revised manuscript, we have realized the little difference between points 2 and 3, and deleted the point 3;

Comment: Results line 105: separate the numbers by ; instead of , to avoid confusion.

Reply: Materials and methods section, line 128-129, page 3 in the revised manuscript, we have separated the numbers by “;” instead of “,”;

Comment: Fig 5: please refer to protein abundance rather than expression.

Reply: Results section, line 317-322, page 12 in the revised manuscript, we have refered to protein abundance in the figure;

Comment: Results line 278/279 please state in which comparison the upregulation was observed.

Reply: Results section, line 303, page 12 in the revised manuscript, we have stated in the comparison;

Comment: Adding an introductory sentence stating what was done and why at the start of each paragraph would make the manuscript easier to follow.

Reply: Discussion section, line 397-398, line 407-408, line 419-422, line 485-486, line 530, page 16-18 in the revised manuscript, we have added the introductory sentence stating what was done and why at the start of each paragraph;

Comment: Discussion line 378: RNAseq does not measure transcription but mRNA levels.

Reply: Discussion section, line 432, page 16 in the revised manuscript, we have replaced “transcription” by “mRNA levels”;

Comment: Discussion lines 424-430: Please explain the purpose of this paragraph. It sounds like a description of the method performed in this work but does this mean the previously performed method is inadequate? What is meant by existing method? The one used in this work?

Reply: Discussion section, line 488, page 17 in the revised manuscript, we have realized the statement of the paragraph is irrelevant, and delected related descriptions.

I hope the revision is satisfactory and please contact me shall you have further questions.

Sincerely,

Tenglong Xie

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript entitled “Exogenous DCPTA increases the tolerance of maize seedlings to PEG-simulated drought by regulating nitrogen metabolism-related enzymes” the authors applied RNA-seq analysis and physiological measurements in order to dissect the effect of DCPTA on drought tolerance in maize. The manuscript is well written and addresses important questions related to climate changes.

Below are my questions and comments addressed to the authors.

First of all, I am a bit surprised from the moderate number of up-/down- DEG in the comparison groups. Do the authors think that this might be related to the depth of the RNA-seq analysis? The maize genome is about 2.3Gb and clean reads between 28-34M could lead to the omission of some transcripts. Also do they use stranded libraries or not? I would be glad to have their opinion on that. The other point is how do they defined 24h of PEG-DCPTA treatment and how many plants they used for RNA-seq experiment. I think this should be mentioned in “Materials and methods”. Concerning RNA-seq: do the authors used replicates for the NGS sequencing or the analysis was performed on a single library for each treatment? I know they use nonparametric NOIseq algorithm but there are 3 packages: NOIseq-real; NOIseq-sim and NOIseqBIO. I think this should also be mentioned to the material and methods. Do the authors deposited the RNA-seq raw data to a public database? Are there other common DEGs in the library pair comparison except the one implicated in N metabolism?

Minor points:

Line 223: “Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG vs. the control; line 224 Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG vs. the control; line 225 Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG+DCPTA vs. PEG “ . I think something is wrong in the table description.

Line 63: “photosynthetic pigment biosynthesis” should not be underlined.

Line 147: “anti-nicotinamide adenine dinucleotide-nitrate reductase” should be just anti-NADH reductase.

Line 140: Which buffer do the authors used for protein extraction? I think this should be mentioned.

Line142: “an appropriate amount of protein was boiled in loading buffer…”. How many µg of proteins were loaded and which buffer was used for loading. Laemmli perhaps?

Author Response

Comment: First of all, I am a bit surprised from the moderate number of up-/down- DEG in the comparison groups. Do the authors think that this might be related to the depth of the RNA-seq analysis? The maize genome is about 2.3Gb and clean reads between 28-34M could lead to the omission of some transcripts. Also do they use stranded libraries or not? I would be glad to have their opinion on that.

Reply: The stranded libraries were not use in this study. As far as we known, the possible reasons of the moderate number of DEGs in the comparison groups are as follows:

Maize seeds. Maize inbred line was used as experimental materials; Experiment condition The experiment was carried out in a controlled growth chamber; Sampling time. The results of this study and our previous study showed that shoot growth had no significant phenotypes change in responsed to PEG and/or DCPTA treatment at 24h after stress (Xie et al. 2017). The shoots of maize seedlings could maintained growth by root growth promotion under early period of PEG treatment, therefore, the differences of shoots in the library pair comparisons of PEG vs. the control was small. Our previous studies showed that DCPTA treatment showed a significant effect on photosynthesis (Xie et al. 2017) and antioxidant system (Xie et al. 2018) from 3^rd day after 15% PEG-6000 treatment, therefore, the differences of seedlings in the library pair comparisons of DCPTA vs. the control and PEG+DCPTA vs. PEG were small at 24h after treatment. Screening criteria for DEG To make the data more reliable and accurate, a higher screening criteria for DEGs was applied, and this may lead to the omission of a mass of transcripts with small difference. Depth of the RNA-seq analysis. RNA-sequencing was performed by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). The qualities of samples were satisfied the needs of RNA-seq (Table 1). In the process of obtaining clean reads, the adaptor sequence and low-quality reads were strictly cut off, and this maybe partly responsible for the moderate number of DEGs.

Table 1 Indexes of sample quality

Comment: The other point is how do they defined 24h of PEG-DCPTA treatment and how many plants they used for RNA-seq experiment. I think this should be mentioned in “Materials and methods”.

Reply: Materials and methods section, line 110-112, page 3 in the revised manuscript, we have mentioned the number of plants for RNA-seq experiment. Our previous study set 5 time point (0, 1, 3, 5 and 7 day after treatment), and the results showed that shoot growth had no significant phenotypes change in responsed to PEG and/or DCPTA treatment at 1 day (Xie et al. 2018). To explore the seedlings response to PEG simulated-drought and/or DCPTA during the shoot had no significant phenotypes change, the study was performed, and the time point of 24h after treatment was selected;

Comment: Concerning RNA-seq: do the authors used replicates for the NGS sequencing or the analysis was performed on a single library for each treatment? I know they use nonparametric NOIseq algorithm but there are 3 packages: NOIseq-real; NOIseq-sim and NOIseqBIO. I think this should also be mentioned to the material and methods.

Reply: Materials and methods section, line 136, page 3 in the revised manuscript, we have mentioned the package used in this study, and the analysis was performed on a single library for each treatment;

Comment: Do the authors deposited the RNA-seq raw data to a public database? Are there other common DEGs in the library pair comparison except the one implicated in N metabolism?

Reply: The RNA-seq raw data of this study had not been deposited to a public database due to our subsequent study, and the data will be deposited to a public database when the work is done. There were common DEGs in the library pair comparisons of PEG vs. the control and PEG+DCPTA vs. PEG, such as XM_008675011.1 and XM_008671868.1 related to glutamatergic synapse ras signaling pathway (ko04014); common DEGs in the library pair comparisons of DCPTA vs. the control and PEG vs. the control, such as XR_559803.1, XM_008664126.1 and XR_559802.1 related to carotenoid biosynthesis (ko00906), XM_008658536.1 and XM_008658537.1 related to aspartate and glutamate metabolism (ko00250); common DEGs in the library pair comparisons of DCPTA vs. the control and PEG+DCPTA vs. PEG, such as NM_001147243.1 and XM_008678619.1 related to cutin, suberine and wax biosynthesis (ko00073), XM_008646063.1 and NM_001134283.1 related to cyanoamino acid metabolism (ko00460). Maybe due to the moderate number of DEGs in the comparison groups, the total number of the common DEGs with pathway annotation in the 3 library pairs was very small, such as NM_001155135.1 related to escherichia coli infection (ko05130).

Comment: Line 223: “Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG vs. the control; line 224 Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG vs. the control; line 225 Table 4. KEGG enrichment analysis of DEGs in the library pair comparison of PEG+DCPTA vs. PEG “ . I think something is wrong in the table description.

Reply: Results section, line 289, page 7 in the revised manuscript, we have checked the typing mistakes, and replaced “PEG” with “DCPTA”;

Comment: Line 63: “photosynthetic pigment biosynthesis” should not be underlined.

Reply: Introduction section, line 75, page 2 in the revised manuscript, we have checked the typing mistakes, and deleted the underline;

Comment: Line 147: “anti-nicotinamide adenine dinucleotide-nitrate reductase” should be just anti-NADH reductase.

Reply: Materials and methods section, line 189, page 4 in the revised manuscript, we have checked the typing mistakes, and replaced “anti-nicotinamide adenine dinucleotide-nitrate reductase” with “anti-NADH reductase”;

Comment: Line 140: Which buffer do the authors used for protein extraction? I think this should be mentioned.

Reply: Materials and methods section, line 180-181, page 4 in the revised manuscript, we have mentioned the buffer used for protein extraction;

Comment: Line142: “an appropriate amount of protein was boiled in loading buffer…”. How many µg of proteins were loaded and which buffer was used for loading. Laemmli perhaps?

Reply: Materials and methods section, line 183-185, page 4 in the revised manuscript, we have mentioned the amount of proteins were loaded and buffer was used for loading.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Most of the points raised were answered adequately and all passages which were unclear in the text are clarified now. Especially the experimental design is nicely described now and the robustness of the analysis becomes more obvious.

I only have two minor points that could be improved: I would move the introductory sentences that were added rather to the results paragraphs corresponding to the experiments and not place them in the discussion.

The second point concerns figure 3: I think it is not clear where the percentages originate from. They are very similar to the numbers of genes. How can one conclude enrichment from these numbers?

Author Response

Comment: I would move the introductory sentences that were added rather to the results paragraphs corresponding to the experiments and not place them in the discussion.

Reply: Results section, line 242-243, 273-274, 300-301, 369-370 and 381, page 6-14 in the revised manuscript, we have moved the introductory sentences to the results section.

Comment: The second point concerns figure 3: I think it is not clear where the percentages originate from. They are very similar to the numbers of genes. How can one conclude enrichment from these numbers?

Reply: Results section, figure 3, line 280-283, page 7 in the revised manuscript, we have realized the statement of the percentages origination is not clear, and amended the explanation. The percentages originated from the comparisons of enriched DEGs population to the background population of all genes in the corresponding GO term performed by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). To identify the major metabolic pathway which DEGs enriched, GO analysis was performed. The GO terms with DEGs enrichment were shown in figure 3. According to the percentages and the number of ordinate in figure 3, the quantity and distribution of the enriched DEGs were shown.