Can Foliar-Applied Omeprazole Improve the Yield, Assimilation, Recovery and Nitrogen Use Efficiency in Bean Plants?

Round 1

Reviewer 1 Report

In the present manuscript, the effect of omeprazole on N assimilation, recovery and N use efficiency parameters for bean plants was evaluated.

How many plants were grown in each pot?

Correct the horizontal axis in all the Figures to make all the letters visible.

Figure 7: correct to applied.

The fact that the obtained yield is below the average yield indicates that the plants were grown under nitrogen deficiency stress. Moreover, the authors do not consider nitrogen fixation in their concept and the fact tha the plants were grown in a substrate and in pots instead of field conditions does not allow to extrapolate the results to real conditions.

Author Response

January 31, 2024

Dear Editor, Ms. Esther Han

Please find enclosed the corrected version of paper entitled: “Can foliar-applied omeprazole improve the yield, assimilation, recovery and nitrogen use efficiency in bean plants?”. (Manuscript ID: 2850348). First, I would like to thank the academic editor and the review team for their constructive and insightful comments, which have greatly improved the quality and clarity of the paper. In addition, we would like to express our gratitude for having had the opportunity to submit the revised paper for reconsideration for publication. Below is a point-by-point response detailing the detailing the observations that were made in the review.

Reviewer 1

Point 1. Two plants were planted in each pot, one destined to be harvested and subsequently destroyed for macerations for nitrate reductase activity, amino acid and sucrose analysis. The remaining plant was harvested, weighed, and then subjected to a drying process to grind it and obtain the mineral fraction of nitrogen and carbon.

Point 2. The font size on the horizontal axis of all graphs was reduced to make it visible.

Point 3. In Figure 7, the dose description was corrected from "Apliedd omeprazole doce" to "Dose of omeprazole applied (µM)".

Having made these corrections, we hope that the manuscript is now suitable for publication in the journal NITROGEN (ISSN: 2504-3129). If you have any further suggestions or questions, please do not hesitate to contact us.

Yours sincerely,

Dr. Esteban Sánchez

Reviewer 2 Report

The manuscript aims to demonstrate how omeprazole treatment on bean plants can improve yield, assimilation, recovery, and nitrogen use efficiency. Although there are similar studies on different plants, the authors fill the gap on bean plants. The objective of the study is clearly explained, but the Introduction should be improved adding much more information and previous studies. Material and methods are adequately described, and the analytic methods are appropriate and do not need further control. Some results are relevant for the readers, but the hypothesis about the reason why there is a negative effect of OMP at high concentrations on yields, NR activity, soluble amino acid, and soluble protein concentration, NUpE and NUE, are not enough. The authors should try to explain, also with the help of any previous works, the clear downward trend observed at the highest doses of OMP.

Furthermore, some points should be addressed prior to further consideration:

Table 1. The table is not necessary if the composition of the Hoagland standard nutrient solution adapted to green bean has been already described in the paper [14] as you have indicated.

Figure 1. This figure is not necessary, the experimental design is clearly described in the text.

Line 101. Adding enzyme entry before E.C. 1.7.1.1 could be better.

Line 264, Line 357-359, Figure 8. You can’t say that the treatments with OMP increases NUtE parameters because the results are not significant!

Line 314. Attention at: “Similarly, Possibly….”

Line 379. Attention at: “ and end…”

Author Response

January 31, 2024

Dear Editor, Ms. Esther Han

Please find enclosed the corrected version of paper entitled: “Can foliar-applied omeprazole improve the yield, assimilation, recovery and nitrogen use efficiency in bean plants?”. (Manuscript ID: 2850348). First, I would like to thank the academic editor and the review team for their constructive and insightful comments, which have greatly improved the quality and clarity of the paper. In addition, we would like to express our gratitude for having had the opportunity to submit the revised paper for reconsideration for publication. Below is a point-by-point response detailing the detailing the observations that were made in the review.

Reviewer 1

Point 1. Two plants were planted in each pot, one destined to be harvested and subsequently destroyed for macerations for nitrate reductase activity, amino acid and sucrose analysis. The remaining plant was harvested, weighed, and then subjected to a drying process to grind it and obtain the mineral fraction of nitrogen and carbon.

Point 2. The font size on the horizontal axis of all graphs was reduced to make it visible.

Point 3. In Figure 7, the dose description was corrected from "Apliedd omeprazole doce" to "Dose of omeprazole applied (µM)".

Reviewer 2

Point 1. As suggested by reviewer 2, more studies about the effects of omeprazole in plants were added. Line 48.

Point 2. Information was added about the downward trend observed with high-dose omeprazole. Line 323.

Point 3. Reviewer 2's suggestion to remove table 2 is accepted.

Point 4. Reviewer 2's suggestion to remove figure 1 is accepted.

Point 5. Added enzyme abbreviation before (E.C. 1.7.1.1). Lines 99 and 212.

Point 6. Following the suggestion of reviewer 2, we removed the sentence stating that treatments with OMP increases NUtE parameters since the results are not significant.

Point 7. “Similarly”, was erased. Line 312.

Point 8. “End products” was changed to just “products”. Line 383.

Reviewer 3

Point 1. “Different letters, for each graphic” was added in each graphic since analyses were made for each graphic.

Point 2. It was clarified that between the control treatment and the OMP 10 treatment there was no significant statistical difference in the induced nitrate reductase enzyme activity. Line 216.

Point 3. It was clarified that between the control treatment and the OMP 100 treatment there was no significant statistical difference in the chlorophyll a. Line 227.

Point 4. The whole paragraph was reformulated to avoid using “slightly increase”. Lines 247 and 260.

Point 5. “yield estimation” was replaced for “yield measurement” Line 294.

Point 6. The sentence was reformulated to establish that only in the case of increasing the dose to 100 µM was a significant decrease obtained. Lines 298-300.

Point 7. “greatest” was replaced for “higher”. Line 331.

Having made these corrections, we hope that the manuscript is now suitable for publication in the journal NITROGEN (ISSN: 2504-3129). If you have any further suggestions or questions, please do not hesitate to contact us.

Yours sincerely,

Dr. Esteban Sánchez

Author Response File: Author Response.docx

Reviewer 3 Report

the figures must be corrected, so that it is clear which treatment each column refers to (X axis). It also should be added, in the legend of the figure, that “Different letters, for each graphic,…” (because the statistic is made for each group, separatelly)

I suggest that you always be careful when there are no significant differences, because if there are not, the treatments are considered not diferente, and it should be clear

Line 219 – when it is written “…the highest NO3- induced NR 219 activity was obtained in the control treatment”, it should be clear that it was not diferente from the OMP10 treatment

Line 228 – “…respect to OMP 10 and OMP 1.”, here should also be mentioned that the OMP100 was not different from the control

Line 301 – the highest yield was obtained in both OMP1 and OMP10 treatments, it just decreased significantly in the OMP100 treatment, and it has to be stated here

Line 323 – I would suggest to use “higher” instead of “greatest”

Author Response

January 31, 2024

Dear Editor, Ms. Esther Han

Please find enclosed the corrected version of paper entitled: “Can foliar-applied omeprazole improve the yield, assimilation, recovery and nitrogen use efficiency in bean plants?”. (Manuscript ID: 2850348). First, I would like to thank the academic editor and the review team for their constructive and insightful comments, which have greatly improved the quality and clarity of the paper. In addition, we would like to express our gratitude for having had the opportunity to submit the revised paper for reconsideration for publication. Below is a point-by-point response detailing the detailing the observations that were made in the review.

Reviewer 1

Point 1. Two plants were planted in each pot, one destined to be harvested and subsequently destroyed for macerations for nitrate reductase activity, amino acid and sucrose analysis. The remaining plant was harvested, weighed, and then subjected to a drying process to grind it and obtain the mineral fraction of nitrogen and carbon.

Point 2. The font size on the horizontal axis of all graphs was reduced to make it visible.

Point 3. In Figure 7, the dose description was corrected from "Apliedd omeprazole doce" to "Dose of omeprazole applied (µM)".

Reviewer 2

Point 1. As suggested by reviewer 2, more studies about the effects of omeprazole in plants were added. Line 48.

Point 2. Information was added about the downward trend observed with high-dose omeprazole. Line 323.

Point 3. Reviewer 2's suggestion to remove table 2 is accepted.

Point 4. Reviewer 2's suggestion to remove figure 1 is accepted.

Point 5. Added enzyme abbreviation before (E.C. 1.7.1.1). Lines 99 and 212.

Point 6. Following the suggestion of reviewer 2, we removed the sentence stating that treatments with OMP increases NUtE parameters since the results are not significant.

Point 7. “Similarly”, was erased. Line 312.

Point 8. “End products” was changed to just “products”. Line 383.

Reviewer 3

Point 1. “Different letters, for each graphic” was added in each graphic since analyses were made for each graphic.

Point 2. It was clarified that between the control treatment and the OMP 10 treatment there was no significant statistical difference in the induced nitrate reductase enzyme activity. Line 216.

Point 3. It was clarified that between the control treatment and the OMP 100 treatment there was no significant statistical difference in the chlorophyll a. Line 227.

Point 4. The whole paragraph was reformulated to avoid using “slightly increase”. Lines 247 and 260.

Point 5. “yield estimation” was replaced for “yield measurement” Line 294.

Point 6. The sentence was reformulated to establish that only in the case of increasing the dose to 100 µM was a significant decrease obtained. Lines 298-300.

Point 7. “greatest” was replaced for “higher”. Line 331.

Having made these corrections, we hope that the manuscript is now suitable for publication in the journal NITROGEN (ISSN: 2504-3129). If you have any further suggestions or questions, please do not hesitate to contact us.

Yours sincerely,

Dr. Esteban Sánchez

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Add details about the total amount of nitrogen that plants received throughout the growing period through nutrient solution.

Why the authors did not use one plant per pot (probably in smaller pots)?

Line 177: check the equation. Is it g² correct? Total biomass is expressed in g.

Equation 8: the term yield is mistakenly used. Yield refers to the biomass per harvested area. Here the authors mean total dry weight. 

Line 221: correct to "statistical".

Line 231: correct to "with".

The authors did not respond to my last comment. As mentioned before, it is difficult to make suggestions for crop production improvements based on pot experiments, especially under the conditions of this experiment (N deficiency, low number of pots, two plants per pot, N-fixation not considered). It is also interesting that although no differences were recorded in total biomass, TNC, TNA, NUtE and recovery of nitrogen between OMP treatments, however there were differences in NUE and NUpE and in yield (e.g. fresh pod weight). The authors should discuss this in more depth.

Moreover, they should extrapolate the obtained results of total biomass, fresh pod weight, the applied amount of nitrogen form pots to field conditions (per hectare). Then the results will be more reasonable. 

See my comments above.

The authors should check their edits in the document.

Author Response

Dear Editor, Ms. Esther Han

Please find enclosed the corrected version of paper entitled: “Can foliar-applied omeprazole improve the yield, assimilation, recovery and nitrogen use efficiency in bean plants?”. (Manuscript ID: 2850348). First, I would like to thank the academic editor and the review team for their constructive and insightful comments, which have greatly improved the quality and clarity of the paper. In addition, we would like to express our gratitude for having had the opportunity to submit the revised paper for reconsideration for publication. Below is a point-by-point response detailing the detailing the observations that were made in the review.

Reviewer 1

Point 1. More details about the amount of nutrient solution applied throughout the growing period and formulas to calculate the nitrogen recovery were added. Lines 182-188.

Point 2. It was decided to use two plants per pot to make the quantification of fresh and dry matter more integrated. In this way, one plant would be left intact to be weighed in its entirety and the other plant would be available for sampling and estimation of enzyme activity, amino acids, proteins. In addition, this ensures that there is enough plant material for the replicates.

Point 3. As described in previous works, the correct NUtE value is expressed as g² DW mg⁻¹ N. Line 173.

Navarro‐León, E., López‐Moreno, F. J., Borda, E., Marín, C., Sierras, N., Blasco, B., & Ruiz, J. M. (2022). Effect of l‐amino acid‐based biostimulants on nitrogen use efficiency (NUE) in lettuce plants. Journal of the Science of Food and Agriculture, 102(15), 7098-7106.

Xu G, Fan X and Miller AJ, Plant nitrogen assimilation and use efficiency. Annu Rev Plant Biol 63: 153–182 (2012).

Point 4. Yield was changed to dry weight of pods to clarify what NUE value means.  Line 177.

Point 5. “Statical” was corrected to “statistical”. Line 217.

Point 6. “Whit” was corrected to "with”. Line 227.

Point 7. Experimentation with plants in pots represents the first step in recommending products to improve crop production. That is, this allows us to have more controlled conditions and to track physiological responses more easily than in field conditions. The completion of this work, together with the reviewers' comments, provides a two-pronged approach. The first is to improve the experimental design in subsequent studies, concentrating on studying the behavior of plants with nitrogen deficiency in more depth, increasing the number of replications, placing only one plant per pot, and taking into account nitrogen fixation. The other way is to transfer the treatments that gave the most favorable results to field conditions and compare the responses when their development occurs under unfavorable conditions (different types of soil, climate, etc.). On the other hand, since there were no marked differences in nitrogen use efficiency parameters between OMP doses, it is worth expanding the range of doses to find out if a dose lower than 1 µM is more efficient or if a dose higher than 100 µM has decreased values. However, the results of this study represent the widening of the knowledge gap about the effects of omeprazole on nitrogen use efficiency for bean plants, making it a valuable study to be replicated and improved.

Having made these corrections, we hope that the manuscript is now suitable for publication in the journal NITROGEN (ISSN: 2504-3129). If you have any further suggestions or questions, please do not hesitate to contact us.

Yours sincerely,

Dr. Esteban Sánchez

Round 3

Reviewer 1 Report

Point 2. You could get the same results if you used only one plant per pot. In this way you can calculate the amount of nitrogen added per pot and per plant and then extrapolate the results to field conditions. By assuming that plants use the soil up to a depth of 20 cm (or the height of the pot) you can calculate the total amount of nitrogen per hectare e.g.  [14.27 mg N (per pot) x 2.000.000 L (the volume of soil of 1 ha)]/9 L (pot volume)=3.2 Kg of N per ha. It seems that this amount is very low. This is probably why no significant dfferences were recorded for most of the parameters. 

Point 3. In the cited reference it is mentioned:

"NUtE was calculated as leaf DW divided by N concentration (g²DW mg⁻¹N)." 

In the original reference of Xu et al. it is mentioned:

“Nitrogen utilization (assimilation) efficiency (NUtE): the fraction of plant-acquired N to be converted to total plant biomass or grain yield. “

If you check the reference of Congreves et al. (2021; https://doi.org/10.3389/fpls.2021.637108) then NUtE is defined as Yield/Plant N.

So, there is a mistake in the equation since yield cannot be expressed in g².

Point 7. The rationale of this work is that the excessive application of N is a problem for agriculture (Lines 13-14, 29-35). However, as mentioned in Point 2, the amounts applied were by no means excessive. So, the tested hypothesis is not relevant.

Author Response

Dear Editor, Ms. Esther Han

Please find enclosed the correct version of paper entitled: “Can foliar-applied omeprazole improve the yield, assimilation, recovery and nitrogen use efficiency in bean plants?” (Manuscript ID: 2850348). First, I would like to thank the academic editor and the review team for their constructive and insightful comments. We would like to give a big thank you to reviewer 1, who through his suggestions and corrections has brought the manuscript to a higher quality version. In addition, we would like to express our gratitude for having had the opportunity to submit the revised paper for reconsideration for publication. Below is a point-by-point response detailing the observations that were made in the review.

Reviewer 1

Point 1. The approach suggested by reviewer 1 to translate the data extracted in the study to field conditions is correct. However, I would like to specify the nitrogen data to carry out the calculations that reviewer 1 gave as an example: The concentration of N per pot is 1588.3938 mg N (per pot) per 2000000 L (volume of soil in one Ha) / 13 L (pot volume). This results in 244.37 kg of N per Ha. Therefore, we consider that this nitrogen value has a physiological response in the plant.

Point 2. The NUtE is calculated by dividing the dry biomass (also called yield or generation of dry mass, whether fruit or aerial mass) by the TNC, which is the total concentration of nitrogen (nitrogen acquired by the plants).

The editor's correction is accepted and it is decided to express the value as suggested by Moll et al. (1982), in its original operation (dry weight / nitrogen acquired by the plant) or g p.s. * mg N^-1.

Point 3. The low efficiency of nitrogen fertilizers is the problem on which the research is based. Therefore, Reviewer 1's correction is accepted and “overapplication of N” is removed from the summary.

Having made these corrections, we hope that the manuscript is now suitable for publication in the journal NITROGEN (ISSN: 2504-3129). If you have any further suggestions or questions, please do not hesitate to contact us.

Yours sincerely,

Dr. Esteban Sánchez