Leaf Nitrogen Traits in Response to Plant Density and Nitrogen Supply in Oilseed Rape
Round 1
Reviewer 1 Report
Agree with the corrections
Author Response
Dear reviewer
We appreciate your positive view on our manuscript. Since one of the reviewers suggested minor changes to our manuscript, we did a final round of corrections to our manuscript (Please see the revised version in the attached file).
With kind regards
Marcelo labra
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This is a revised manuscript that has been improved and the authors have most probably taken into account the reviewers’ comments, as I did have access only to a marked pdf-document and no covering letter that would show the changes made.
Some parts still need revision:
L20: The Abstract should be self-explanatory, so abbreviations in Abstract should be written out: change BBCH62 to something that can be understood without reading the whole article. Also only the most important, and thus always significant, results should be reported in abstract, so the words “significant” and the p-values should be removed.
L63-65: Should be removed, as it is really hard to decipher from the original article the actual result, as the statistical analysis seems to b not properly done, there. The explanation of authors does not clarify the sentence, either.
L271-278: I still disagree about reporting the results the way the authors do, i.e. they merge the results for the plant height so that it is impossible for the readers to see the original data and see by their own eyes if there is indeed no interaction between the treatments. The explanation by drawing the data based on statistical analysis is poor and the statistical test results can still be shown in the figure even if the readers are shown the original data. Plant height was affected by the density and the N-supply, but no interaction means, that these treatments affected the height growth in the same way: So the plants were taller with lower density in both N-treatment levels and in higher N-supply irrespective of density. This should be shown in the figure 2a. Same comment applies to fig. 4b.
There are no comments how the discussion was changed according to my previous comment to improve it. In addition, there are no replies to the comment on citing papers with crops rather than woody evergreens species. At least there seems to be no changes in the reference list.
Minor comments:
L57: has been found to be negatively correlated
L442: something missing?
Author Response
Dear reviewer
Thank you very much for your comments. The new version of the manuscript was revised based on your suggestions. Please find below our responses to your comments.
Response to reviewer #2:
This is a revised manuscript that has been improved and the authors have most probably taken into account the reviewers’ comments, as I did have access only to a marked pdf-document and no covering letter that would show the changes made.
We appreciate the positive view by the reviewer on our manuscript. In the revised version introduction, material and methods, results and discussion were rewritten according to your comments and suggestions in the previous and current rounds of revision (please, see this revised version in the attachment).
Some parts still need revision:
L20: The Abstract should be self-explanatory, so abbreviations in Abstract should be written out: change BBCH62 to something that can be understood without reading the whole article. Also only the most important, and thus always significant, results should be reported in abstract, so the words “significant” and the p-values should be removed.
In line 20 of this revised manuscript the sentence “…N content of selected leaves were measured at BBCH 62”, was replaced by “…N content of selected leaves were measured at 20% of flowers on main raceme open”
In line 21 of the revised manuscript the sentence “…the interaction between N supply and plant population density significantly (P<0.05) altered leaf N content per area”, was replaced by “…the interaction between N supply and plant population density altered leaf N content per area”.
In line 23 the sentence “(P=0.003)” was removed.
L63-65: Should be removed, as it is really hard to decipher from the original article the actual result, as the statistical analysis seems to b not properly done, there. The explanation of authors does not clarify the sentence, either.
In the revised version the sentence “From the results of this last study, it is possible to hypothesize an interaction between N supply and plant population density, but this was not considered in their analysis” was removed as suggested by the reviewer. Please see line 58 of this revised version.
L271-278: I still disagree about reporting the results the way the authors do, i.e. they merge the results for the plant height so that it is impossible for the readers to see the original data and see by their own eyes if there is indeed no interaction between the treatments. The explanation by drawing the data based on statistical analysis is poor and the statistical test results can still be shown in the figure even if the readers are shown the original data. Plant height was affected by the density and the N-supply, but no interaction means, that these treatments affected the height growth in the same way: So the plants were taller with lower density in both N-treatment levels and in higher N-supply irrespective of density. This should be shown in the figure 2a. Same comment applies to fig. 4b.
Figs 2a and 4b were modified as suggested by the reviewer. In this revised version the mean and standard error of each treatment were included to facilitate the analysis of the readers. The significance (P value) of the effect of both N supply and plant population density were also added in Figs 2a and 4b.
Minor comments:
L57: has been found to be negatively correlated.
The verb “to be” was added to the sentence (please see line 54 in the revised version)
L442: something missing?
This was corrected and the revised version as: “Discrepancies among these studies could be due to the fact that effects of N and light on LMA are hidden by their interaction” (please see line 430 of the revised version)
There are no comments how the discussion was changed according to my previous comment to improve it. In addition, there are no replies to the comment on citing papers with crops rather than woody evergreens species. At least there seems to be no changes in the reference list.
We regret that the reviewer did not get access to the cover letter and response to the comments done in the previous round of revision. Indeed, the revised version considered most of the improvements suggested by the reviewer. Please see details below:
Discussion
Line 356. “…of the evaluated leaves...” irrelevant.
This was erased as suggested by the reviewer.
Line 368. Fig 8. Can not be in a discussion part. Makes part of the results.... In the revised version,
Fig. 8 was moved to results.
Line 375, and line 378 “…higher plant height? English please.
This was replaced by “…taller plants”
Line 375. Did you measure the plant stem diameter to confirm this... thinner?
It was not included in this manuscript, but it is supported by the literature. Xue, J.; Gou, L.; Zhao, Y.S.; Yao, M.N.; Yao, H.S.; Tian, J.S.; Zhang, W.F. Effects of light intensity within the canopy on maize lodging. Field Crops Res 2016, 188, 133-141, doi:10.1016/j.fcr.2016.01.003.
To avoid confusing interpretations by readers, the following comment was added in line 385:… “(not measured in the current experiment)”
Line 377. increase in lodging risk [53], What is this? Explain.
Lodging is the bending over of the stems near ground level of grain crops, which makes them very difficult to harvest, and can dramatically reduce yield. Lodging in cereals is often a result of the combined effects of inadequate standing power of the crop, and conditions such as rain, wind, hail, topography, soil, previous crop, and others. This definition can be found in: Berry, Pete M. (5 April 2018). "Lodging Resistance cereal lodging resistance in Cereals cereal". Encyclopedia of Sustainability Science and Technology. Springer, New York, NY. pp. 6201–6216. doi:10.1007/978-1-4419-0851-3_228 ISBN 978-0-387-89469-0.
Since lodging in oilseed rape is a concept widely known in crop science, we believe further explanations are not needed.
Line 379 Explain in a fisiological way why it has happened.
This is explained (please see line 391) in the following sentence: “This behaviour is the result of an intensive intra-specific competition for resources (i.e., light or nutrients), at developmental stages when the plant requirements are higher than the resources available to satisfy such requirements, as has been demonstrated in sunflower [57] and now in oilseed rape. Thus, at conventional plant densities (e.g. 50 plants m-2), increasing plant population density leads (e.g. 150 plants m-2) to taller plants, but there is a critical level above which further increases in plant population density will cause a reduction in plant height, due to a shortage in resources available for plant C assimilation, causing a reduction in photoassimilates available for plant growth.”
Line 384. “Thus, at conventional plant densities…” What is this? Explain in your case, if appliable.
In the revised version this was better explained as follows: ”…Thus, at conventional plant densities (e.g. 50 plants m-2), increasing plant population density (e.g. 150 plants m-2) leads”
Line 388. “…while a delay in leaf appearance was evident only in the final leaves evaluated…” Why? Explain better.
This was better explained in the following sentence: “…while a delay in leaf appearance was evident only in the final most apical leaves evaluated.”
Line 390. What are optimal conditions? Explain.
In the revised version “…Under optimal conditions” was replaced by “When soil N status exceeds the plant N requirements”
Line 391. “Thus, N is absorbed, assimilated and distributed in the plant to maximize photosynthesis” Only? I am not sure. What other activities are conditioned by N availability in the plant?.
Many other plant activities are driven by the N absorbed, but most of them takes place during the reproductive stage. This paper focus on the reproductive stage when leaf photosynthesis is the main physiological process affected by N uptake.
Line 406. Picea is also strange tree, no leaves present as you have in oilseed rape. Chose another exmple”.
In the revised version, reference of line 406 (Picea abies) was removed from the revised manuscript as suggested by the reviewer.
Line 414-416. Phenological stages are iqual in rape plants. The problem is their duration. The problem may be also in a period of year when plants are grown or even variety.
This was considered and added in the following sentence “… and the moment of the season in at which these phenological stages occur. This is important when comparing winter and spring oilseed rape or different varieties.”
Line 422.
“…as observed in our study” was added.
Line 428. “In our work, An was strongly correlated with Narea in an asymptotical relationship” Why? Explain please using physiology examples.
Since the asymptotical relationship between An and Narea has been observed and discussed in previous studies (for example Archontoulis et al. 2012 or Trapani & Hall 1996. See references below), but it is not the central objective of the current study, it was not further discussed.
Archontoulis, S.V.; Yin, X.; Vos, J.; Danalatos, N.G.; Struik, P.C. Leaf photosynthesis and respiration of three 589 bioenergy crops in relation to temperature and leaf nitrogen: how conserved are biochemical model 590 parameters among crop species? J Exp Bot 2012, 63, 895-911, doi:10.1093/Jxb/Err321. 591 48.
Trapani, N.; Hall, A.J. Effects of leaf position and nitrogen supply on the expansion of leaves of field grown 592 sunflower (Helianthus annuus L). Plant Soil 1996, 184, 331-340, doi:10.1007/Bf00010462. 593
Line 433-435. Explain with citations.
This sentence was modified and the reference below was added:
Sorin, C.; Leport, L.; Cambert, M.; Bouchereau, A.; Mariette, F.; Musse, M. Nitrogen deficiency impacts on leaf cell and tissue structure with consequences for senescence associated processes in Brassica napus. Bot Stud 2016, 57, doi:ARTN 11 10.1186/s40529-016-0125-y.
Line 444. “RUE”. What is this? Did you define somewhere?
The acronym RUE was replaced by “radiation-use efficiency”
Line 459. something is missing here. Not clear.
The sentence was modified as follows: “and scaled up from the leaf to biological organizational levels relevant for ecophysiology and agronomy (i.e., population level)”
Supplementary materials:
The effect of the main factors, and interactions between factors were included as supplementary materials in the section “appendix” as suggested by the one of the reviewers. This was done for the following traits: Plant height, Leaf appearance, leaf area, N content per leaf, Narea, LMA, Nmass, N uptake m2 and per plant.
Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
This manuscript has to be rewritten in some parts, mainlu Introduction, results and discussion.
Introduction is not very clear.
MM part is the best, but needs some more improvements.
Results, should be elaborated better.
Discussion, and conclusions, need some justifications of the results that are not only related with the results, but also some study of physiology. Like it is can not be accepted.
You should follow my comments and please make the manuscript better.
Comments for author File: Comments.pdf
Reviewer 2 Report
This research utilises an experimental setup with two different nitrogen levels and plant densities with a crop plant oilseed rape. The primary objective is to find out the nitrogen distribution within the plant in these different conditions. The main findings of the paper are that both plant density and N supply, alone and combined, affect plant architecture, leaf properties and the N distribution within the plants in different ways.
Overall, the manuscript is well written. The experimental setup is sound, and the methods used are well described. Some figures and tables are unfortunately on two different pages, but the layout can be easily corrected. However, some corrections are needed before I can recommend the manuscript it for publication.
The statistical analyses seem to be valid, but the all the test results should be presented more clearly, for example in a table (as a supplement, if cannot be part of the MS). Table 2 presents the effects clearly, but this data are missing from Figs. 3&4. Only the interaction is told, but not the main effects of the N and density results. Figures 2a and 4b should present the results separately for each treatment combination instead of combining them for N and density, although no statistical interaction was found.
Discussion should answer the research questions posed is the introduction in more detail. The first part of discussion about plant level effects is now concentrating more on the N supply effect than the density or their interaction, although the interaction was observed for example for N uptake (Fig.7b). The leaf level effects should not repeat the results, but tell about the observed effects (for example lines 404-405: what was the response of LMA?).
The references citing trees and especially evergreen conifers are not valid comparison points to an annual crop when the resource allocation is discussed (for example line 406) and the references citing these should be replaced with citations to annual species.
