Effects of Nitrogen and Water Deficiency on Agronomic Properties, Root Characteristics and Expression of Related Genes in Soybean

Round 1

Reviewer 1 Report

Dear authors,

The subject of the revised article is current, original, and presents the results of a complex and multidisciplinary scientific study. Knowing the tolerance of the world's oilseed crop to the main limiting factors such as drought and nutrient supply is of great importance in agriculture.

This paper is composed of Abstract, Introduction, Materials and methods, Data collection, Results, Discussion Conclusions and References.

The selection of "Materials and methods" is correct. The choice for experiments of non-genetically modified soybean genotypes is to be appreciated. It is worth noting the choice of classical study methods and those of modern genetics. The number of 16 variants adopted is adequate. If the degree of freedom for estimating the standard error is adequate, because the statistical processing was performed with specialized and high-performance software (R package, version 3.4.1 [28]) the results are centralized in Table 4 & Figure 1 .... Figure 7 is edifying.

The description of tables and figures is legible and understandable. The results of these studies may be important in breeding programs and in agricultural practice.

I would recommend that based on the obtained results  to make some suggestions regarding the differentiated application of nitrogen fertilizers to soybean genotypes according to the climatic characteristics of the areas where they are cultivated.

The article presents 41 bibliographic sources representative of the topic studied and the validation of the obtained results.

Author Response

Dear Reviewer,

We are very thankful for taking the time to evaluate our manuscript and we would like to thank you for the comments based on that we can improve the overall quality of the manuscript.

We agree with the reviewer that the subject of this article is current. The reviewer emphasized that the experimental design was appropriate and the statistical processing was adequate.

The reviewer recommended making some practical suggestion regarding the nitrogen fertilization of the soybean, therefore we completed the Conclusions. But it must be taken into consideration that we analysed the responses of genotypes under limited nitrogen supply and aimed to detect genotypic variations, but the model study is not suitable to make an exact and general recommendation for soybean production under real field conditions. Besides the demand of the harvested cultivar, the capabilities of the field must be always taken into consideration by the planning of the fertilization and other agronomic traits as well.

Reviewer 2 Report

Authors have tried to investigate the combined impact of drought stress and nitrogen deficiency in four different varieties of soybean plants. Nitrogen deficiency and its impact on plant growth and development is a well-known phenomenon. In this current format of the manuscript, the experimental setups are not so promising. Here are few concerns about the experimental design needed to be addressed in order to improve their quality of findings. It would be better if authors could have done drought stress and nitrogen deficiency experiments at different levels of water and nitrogen concentration instead of taking only two levels, 100%, and 50%.  For the complete gene expression analysis of nitrogen assimilation-related genes, authors could have done gene expression analysis in all the selected varieties of soya bean plants at different time points and in various combinations of drought stress and nitrogen deficiency.

Author Response

Dear Reviewer,

We agree with the general comments of the reviewer that “the nitrogen deficiency and its impact on plant growth and development is a well-known phenomenon” because it is widely studied by various field crops as well. But the effects of the nitrogen-limited environment in a combination with soybean is not intensively studied. Furthermore, generally, the related studies are focusing on the aboveground biomass and yield parameters but we exactly measured the effects of the tested factors on the root development of the plants as well. The root system and its development under non-favourable conditions would be a key factor in plant stress adaptability, therefore our study has originality. The experimental design can be always more complex but even a multifactorial experimental layout can be disadvantageous because it could mask the effects of the individual factors. In our experiment, we focused on the effects of the nitrogen supply and the water deficiency on plant production and root parameters and the reactions of four important genotypes were tested. We agree with the reviewer that even diversified nitrogen and water supply levels could be tested but based on the results of this trial, selection of genotypes with different reactions to drought and nitrogen fertilization rate become possible for a further, more diversified study.

“For the complete gene expression analysis of nitrogen assimilation-related genes, authors could have done gene expression analysis in all the selected varieties of soya bean plants at different time points and in various combinations of drought stress and nitrogen deficiency.”

Gene expression analysis is an expensive and labour-demanding process, the complete gene expression analysis of all, nitrogen assimilation related genes were not possible in our experiment, and that is why we did not analyse all varieties, but the selected two, differently reacting ones.

Reviewer 3 Report

The manuscript entitled “Root development and abiotic stress tolerance of four soybean varieties under simulated drought conditions in a combination with nitrogen deficiency” aimed to characterize the response of soybean to the N and water stress, and check which cultivar growing best under the unfavorable environments. The topic is broad of interest, and the manuscript was well written. However, the below issues should be addressed.

The most important physiological characteristics of soybean for farmers are the total biomass and grain yield as shown in Table 4. It seems that Bagera is the most tolerant cultivar under the N and drought stress, with the relatively highest biomass and yield. BUT the authors concluded Pannónia Kincse is the best one.

The title did not totally include what they presented in the manuscript. They compared the characteristics of biomass, yield, roots, leaves, and gene expression, but only highlight the root development in the title, which is not inclusive.

The abbreviations that appeared for the first time in the abstract should be provided by the full name.

The authors mentioned the ongoing climate change will cause challenges to soybeans, but only did the analysis on the drought stress. The temperature increase might be the more important one which could cause yield loss of about 4% by each degree, see reference Zhao, C., et al. (2017). Temperature increase reduces global yields of major crops in four independent estimates. Proceedings of the National Academy of Sciences, 114(35), 9326-9331. They should explain why they only analyze the drought stress or include parts of discussion for the outlook in the manuscript.  

Interestingly, they measured some indexes of gene expression. But my feeling is they did not absolutely connect them with the biomass and yield changes. In most cases, the trend of gene and biomass index under different stress was not similar, which is hard to explain why the yield is different and conclude the best cultivar to the stress environment.

The colors of bars/lines should be kept always same for the specific treatment across the manuscript, rather than too many different colors.

Author Response

Dear Reviewer,

We are very thankful for taking the time to evaluate our manuscript and we would like to thank you for the comments based on that we can improve the overall quality of the manuscript.

We agree with the reviewer that the examined topic is broad of interest, and thank you for your positive opinion about the manuscript.

 

“The most important physiological characteristics of soybean for farmers are the total biomass and grain yield as shown in Table 4. It seems that Bagera is the most tolerant cultivar under the N and drought stress, with the relatively highest biomass and yield. BUT the authors concluded Pannónia Kincse is the best one.”

Pannónia Kincse had significantly higher aboveground biomass under control watering in both nitrogen treatment, only under combined stress the biomass of Bagera was higher. The yield data are visualized in Figure 1. It can be concluded that no significant differences were between Bagera and Pannonia Kincse while the responses of Pannonia Kincse was better to the nitrogen-limited environment, its grain yield was significantly higher than that of Bagera. The two nitrogen treatments cannot compare statistically (ANOVA showed any significant effects of the N treatments) but the yield of Bagera decreased when the nitrogen supply was limited. This trend was not detectable in Pannónia Kincse.

 

“The title did not totally include what they presented in the manuscript. They compared the characteristics of biomass, yield, roots, leaves, and gene expression, but only highlight the root development in the title, which is not inclusive.”

We agree with the reviewer’s suggestions. The experimental design was complex and various parameters were measured. Therefore the title has been revised and modified. (Effects of nitrogen and water deficiency on agronomic properties, root characteristics and expression of related genes in soybean)

 

“The abbreviations that appeared for the first time in the abstract should be provided by the full name.”

Abbreviations were explained even in the Abstract

 

“The authors mentioned the ongoing climate change will cause challenges to soybeans, but only did the analysis on the drought stress. The temperature increase might be the more important one which could cause yield loss of about 4% by each degree, see reference Zhao, C., et al. (2017). Temperature increase reduces global yields of major crops in four independent estimates. Proceedings of the National Academy of Sciences, 114(35), 9326-9331. They should explain why they only analyze the drought stress or include parts of discussion for the outlook in the manuscript.”

The Carpathian Basin located on the northern boundary of the soybean cropping area where this plant could be harvested efficiently. The most important limiting factor in this region is the temperature. Typically, early ripening genotypes can be grown, therefore one of the positive consequence of climate change in this region could be that the soybean production becomes safer. Therefore, the effects of this factor, which can an important stressor in many parts of the world was not involved in the present study.

 

“Interestingly, they measured some indexes of gene expression. But my feeling is they did not absolutely connect them with the biomass and yield changes. In most cases, the trend of gene and biomass index under different stress was not similar, which is hard to explain why the yield is different and conclude the best cultivar to the stress environment.”

Acclimation to the changing environment also involves the adjustment of expression levels of various regulating genes, and earlier results also suggested that certain genes could be differentially expressed among the N-tolerant and N-sensitive varieties under N-limited conditions. In order to better understand the main mechanisms regulating stress acclimation and/or nitrogen assimilation related processes, certain genes playing a key role in these processes were chosen. These included the Nitrate reductase, which is involved in the first step of nitrate assimilation in plants; the GmTP55 is an aldehyde dehydrogenase gene, which encodes an antiquitin-like soybean protein involved in drought tolerance; the Cyp707 gene, which encodes an ABA 8′-hydroxylase-induced protein (ABA is a key hormone in drought acclimation), and a DREB gene encoding an ABA-dependent transcription factor. Our results demonstrated the effects of certain treatments and differences in stress responses between the genotypes. However, since the molecular mechanisms are complex, especially under combined stress conditions, a direct relationship between the gene expression changes and the yield components could not be expected. What we expected, and what we got was to get clearer pictures about these molecular mechanisms in different genotypes. For its better understand, the conclusion has been completed with the following text: “Since no direct correlation was found between the expression levels of each gene and the yield parameters, further studies are needed to better understand the detailed stress-regulating mechanisms.”

“The colors of bars/lines should be kept always same for the specific treatment across the manuscript, rather than too many different colors.”

The colour-coding has been harmonized across the manuscript except Figure 1 because in these graphs only two treatments were presented.

Round 2

Reviewer 2 Report

Authors have addressed all the concerns

Author Response

Dear Reviewer,

Thank you very much for taking the time to evaluate our manuscript.

We thankfully appreciate the positive opinion of the Reviewer about the improved version of our manuscript.

Reviewer 3 Report

Unfortunately, the authors did not improve too much their manuscript in this round.

The research is to explore which cultivar is most tolerant to the drought and N deficit, rather than to choose which one has the highest yield. They did not use a scientific method to get a conclusion. The cultivar of Pannónia Kincse shows the highest absolute biomass and yield, but the relative changes under stress treatment is not least. The other issue is that the yield with N stress is surprisingly higher than that under ambient environment, which means the less N is helpful to soybean. The result is out of our common knowledges.

The abbreviation of BBCH13 was still not given the full name in the abstract.

I suggest they should explain why they only analyzed the drought treatment, but I did not find any new texts in this version.

The new interest to the section of gene expression was obliterated, as they did not build a clear bridge between the macroscopic phenomenon and microscopic explanations, with respect to the climate change effect. The new version of MS did not show any discussions on this.

Author Response

Dear Reviewer,

We are very thankful for taking the time to evaluate our manuscript and we would like to thank you for the comments based on that we can improve the overall quality of the manuscript.

We revised deeply the manuscript considering your instructions. We completed the Introduction, the Discussion as well as the Conclusions chapter.

Responses the Reviewer’s comments:

• The research is to explore which cultivar is most tolerant to the drought and N deficit, rather than to choose which one has the highest yield.

The aims of the study were introduced at the end of the Introduction:

1. How does N supply affect the main agronomic parameters of soybean plants?

Here the basic hypothesis was that the nitrogen supply influences the agronomic and phenological properties of the genotypes. This was confirmed.

2. How does water limitation affect the responses of the plants to different N levels?

Here, the basic hypothesis was that the genotypes differ in their responses to the nitrogen supply. The genotype-specific responses are confirmed in various studies to the water-limited environment, the elevated CO₂, etc. and there is variety-specific variability in the optimum rate of nitrogen supply.

3. How do various soybean varieties differ in their abiotic stress tolerance?

Here, we aimed to detect the differences in the adaptation capacities of the examined genotypes to non-favourable conditions. As a result of the study, we confirmed the differences.

 

• They did not use a scientific method to get a conclusion.

All the described changes and differences were determined based on the outcomes of the statistical analysis which consists of an analysis of variance and a post-hoc test for comparing means (if it was relevant). The experimental design, the conducted measurements and the data processing were appropriate from a scientific point of view.

• The cultivar of Pannónia Kincse shows the highest absolute biomass and yield, but the relative changes under stress treatment is not least.

The reviewer’s previous comment in this regard was: “The most important physiological characteristics of soybean for farmers are the total biomass and grain yield as shown in Table 4. It seems that Bagera is the most tolerant cultivar under the N and drought stress, with the relatively highest biomass and yield. BUT the authors concluded Pannónia Kincse is the best one.”

We are thankful to the reviewer for this comment and based on the suggestions we revised and modified the Conclusions.

There were no differences between the yield production of Bagera and Pannónia Kincse under 100% nitrogen supply either in the well-watered or in the drought-stressed treatments. Under limited nitrogen supply, the grain production of Pannóni Kincse was significantly higher than that of Bagera. But it is true, that the ranking changed under limited water availability. We agree with the reviewer, maybe Bagera would be a better choice for gene expression analysis when we would take into consideration the reactions of the varieties to drought but the reactions to nitrogen limitation seemed to be more important from the point of sustainability.

We confirm in the corresponding text that „Bagera produced the highest yield under limited watering in each nitrogen treatment.”. (Line 203.)

• The other issue is that the yield with N stress is surprisingly higher than that under ambient environment, which means the less N is helpful to soybean. The result is out of our common knowledges.

In our experiment, we tested the effects of drought stress and limited nitrogen supply on soybean varieties. In terms of the drought, the treatment was exactly induced stress conditions because the water supply was calculated based on the water-holding capacity of the applied culture media. The half-strength Hoagland solution is widely applied to supply the nutrients for plants.

The reduction of the nitrogen concentration in the solution could lead to stress, but because of the differences in the nitrogen uptake capability of the genotypes, it could be that the treatment is not enough strong to induce stress conditions. Therefore, we use the „drought stress” and „limited nitrogen supply” expressions in the manuscript. The treatments had been started at the early stage of development, which could induce acclimation of genotypes having better adaptation capacities. The special importance of the nitrogen supply in soybean production was confirmed by previous publication as well and that reference has been cited in the revised version of the manuscript.

It is true, that the grain yield value seems to be higher under limited nitrogen supply, but the effect of the nitrogen supply on grain yield was not statistically significant. Therefore, comparing the two values would be not relevant from a scientific point of view. We anywhere stated in the manuscript that the yield of Pannonia Kincse would be higher under limited nitrogen supply than in the control treatment.

 

• The abbreviation of BBCH13 was still not given the full name in the abstract.

We agree with the reviewer because all abbreviations must be explained by the first mention in the text of the manuscript as well as in the Abstract. But the BBCH scale is widely used to determine the exact growth stages of various plant species. The acronym is originated from the German expression „"Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie". The Reviewer should take into consideration that the authors have to shortly introduce the aims of the study, the applied methodologies and the results in the Abstract, which extension is limited (maximum 200 words), therefore, explaining such a well-known acronym seem to be redundant here. However, we rephrase that sentence and we use the „3 leaves stage” instead of the more exact BBCH 13.

 

• I suggest they should explain why they only analyzed the drought treatment, but I did not find any new texts in this version.

The introduction has been completed and we emphasized why our study focuses on the effects of drought stress and why not the potential impacts of the high temperature had been quantified.

 

The new interest to the section of gene expression was obliterated, as they did not build a clear bridge between the macroscopic phenomenon and microscopic explanations, with respect to the climate change effect. The new version of MS did not show any discussions on this."

The Discussion has been completed with the evaluation of the gene expressions in terms of the phenological and yield parameters. But the reviewer should take into consideration that we clearly defined at the end of the Conclusions that, „no direct correlation was found between the expression levels of each gene and the yield parameters, further studies are needed to better understand the detailed stress-regulating mechanisms.”