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Dynamic Effects of Sodium Selenite on the Rhizospheric Microenvironment, Growth, and Antioxidative Responses of Wheat (Triticum aestivum L.)

Agronomy 2025, 15(6), 1427; https://doi.org/10.3390/agronomy15061427

by Fang Qin, Han Zhang, Feiyan Zhang, Xiangrui Zhu, Hongji Li and Yuefeng Xu^*

Reviewer 1:

Bourlaye Fofana

Reviewer 2:

Mauro Wagner Oliveira

Reviewer 3: Anonymous

Reviewer 4: Anonymous

Agronomy 2025, 15(6), 1427; https://doi.org/10.3390/agronomy15061427

Submission received: 29 April 2025 / Revised: 31 May 2025 / Accepted: 9 June 2025 / Published: 11 June 2025

(This article belongs to the Section Soil and Plant Nutrition)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Manuscript Agronomy -3643415-peer-review v1, entitled

Dynamic effects of sodium selenite on rhizospheric microenvi-2ronment, growth, and antioxidative responses of wheat (Triti-3cum aestivum L.) by Fang Qin et al addresses a relevant topic and may be of interest to readers and peers in the field. The authors used Se supplementation in potting experiments. Whereas the data collection and acquisitions appear to be well conducted, the experimental design from which the data were collected were not properly described and lack clarity. The reported appeared to be confirmatory and their originality not clearly aid out As currently presented, the manuscript lacks important methodological details and precision in design and methods to support some data. Therefore, I would recommend rejection of this manuscript in its current form.

Specific concerns can be found below.

Major concerns:

Abstract

The abstract to be rewritten in light of proper experimental design/description and literature review to report original data drawn from the experiment.

Introduction

Lack of full literature review on Se metabolism and translocation in plants
English quality to be improved throughout the text
Improper full literature review to highlight potential areas of originality

Methodology

Lack of details on whay both rhizobags and pots were both used:

why was the rhizobags used if no seeds planted in them but in the pots?
In the design described, has there been any pots not planted with wheat as control? No description/detail is provided
There is no indication in the M&M that the rhizobags were planted with wheat. why would they be considered as rhizosphere soil?
A single experiment appeared to be conducted although 3 reps/unit reported

Lack of details on what was used as control

What was used as control without root?
What was used as contro without or supplemented organic matter

Result section

As raised above in the M&M section, the result section mentions rhizosphere, non-rhizosphere and controls. But there is no indication in the M&M what they refer to and where do rhizosphere, non-rhizosphere and controls come from.
Data on the figures are well presented, but their contents and validy are contingent to the methodology they derive from. – which is not clear to this reviewer

Discussion

A proper literature review would allow the authors to know that there are differences between plants that are Se accumulators and non-accumulators, and that foliar Se application would also allow more accumulation in the leaf than in the root. It would also allow proper discussion of the current findings to layout the points of originality. Increased Se accumulation with increased Se application doses is no not new.
Do the authors refer to Se accumulation and forms or Se accumulation and morphology? What do they stand by morphology?
Are they comparing sodium selenate and sodium selenite?

The discussions do not clearly point out what is original and what is confirmtory.

Conclusions.

In the conclusion, it is said"on the rhizosphere microenviron-460ment, growth, and antioxidant responses of wheat. ". The conclusion should reflect de described experimental design
Without a proper description of control pots, it is not appropriate to claim some conclusions such as “The results showed that the rhizosphere pH and dissolved organic carbon in the soil solution were higher than those of the non-rhizosphere soil solution, and the total Se and inorganic Se in the soil solu-463tion increased with the increase in Se application concentration” –

what was considered in the experiment as pots with and without rhizosphere?
What was the control pots to which the conclusions are drawn for Se contribution to DOC concentration? No where in the text, it is explained what DOC stands for..

Comments on the Quality of English Language

Quality of English should be improved.

Author Response

Dear Reviewers,

The manuscript of agronomy-3643415 has been carefully revised according to your opinions. We appreciate the detailed and useful comments and suggestions from you. These suggestions are very helpful for us to improve the manuscript. Our point-to-point responses to the comments and suggestions are listed as below.

The Manuscript Agronomy -3643415-peer-review v1, entitled Dynamic effects of sodium selenite on the rhizospheric microenvironment, growth, and antioxidative responses of wheat (Triticum aestivum L.) by Fang Qin et al addresses a relevant topic and may be of interest to readers and peers in the field. The authors used Se supplementation in potting experiments. Whereas the data collection and acquisitions appear to be well conducted, the experimental design from which the data were collected were not properly described and lack clarity. The reported appeared to be confirmatory and their originality not clearly aid out As currently presented, the manuscript lacks important methodological details and precision in design and methods to support some data. Therefore, I would recommend rejection of this manuscript in its current form.

Specific concerns can be found below.

No.1.1 The abstract to be rewritten in light of proper experimental design/description and literature review to report original data drawn from the experiment.

Response: Thank you for your kind suggestion. We have revised the manuscript abstract and touched up the grammatical usage through a professional company.

No.1.2 Lack of full literature review on Se metabolism and translocation in plants

English quality to be improved throughout the text

Improper full literature review to highlight potential areas of originality

Response: Thanks for your comment. Considering that this study did not investigate selenium metabolism and conversion within plants, this topic was not mentioned in the introduction. However, we will pay attention to this area in future research. Regarding the quality of the english, we have already used the proofreading services on the MDPI platform.

No.1.3 why was the rhizobags used if no seeds planted in them but in the pots?

In the design described, has there been any pots not planted with wheat as control? No description/detail is provided

There is no indication in the M&M that the rhizobags were planted with wheat. why would they be considered as rhizosphere soil?

A single experiment appeared to be conducted although 3 reps/unit reported

Lack of details on what was used as control

What was used as control without root?

What was used as contro without or supplemented organic matter.

Response: Thanks for your suggestions. We accept the referee’s suggestion and have supplemented the description of the root bag trial design in the revised manuscript as "Where we planted wheat is planted in root bags and then placed the root bags in pots, the reason for the use of the root bags was to differentiate between rhizosphere and non-rhizosphere soils." In this study, we set up five exogenous Se gradients (0.0, 1.0, 2.5, 5.0, and 10 mg Se kg^-1). To ensure the proper conduct of the experiment, each treatment was prepared with fifteen replications and each treatment selected four potted plants with uniform and good growth at 4, 6, and 8 weeks after planting. Our control treatment was an exogenous Se gradient of 0 mg Se kg^-1.

No.1.4 As raised above in the M&M section, the result section mentions rhizosphere, non-rhizosphere and controls. But there is no indication in the M&M what they refer to and where do rhizosphere, non-rhizosphere and controls come from.

Data on the figures are well presented, but their contents and validy are contingent to the methodology they derive from. – which is not clear to this reviewer.

Response: Thank you very much for your advice. We accept the referee’s suggestion and have supplemented the description of the root bag trial design in the revised manuscript as "Where we planted wheat is planted in root bags and then placed the root bags in pots, the reason for the use of the root bags was to differentiate between rhizosphere and non-rhizosphere soils." In this study, soil in the rhizobags was collected and regarded as rhizosphere soil. The soil in the pot 5 mm away from the rhizobag was collected, mixed, and considered to be non-rhizosphere soil. Our control treatment was an exogenous Se gradient of 0 mg Se kg^-1.

No.1.5 A proper literature review would allow the authors to know that there are differences between plants that are Se accumulators and non-accumulators, and that foliar Se application would also allow more accumulation in the leaf than in the root. It would also allow proper discussion of the current findings to layout the points of originality. Increased Se accumulation with increased Se application doses is no not new.

Do the authors refer to Se accumulation and forms or Se accumulation and morphology? What do they stand by morphology?

Are they comparing sodium selenate and sodium selenite?

The discussions do not clearly point out what is original and what is confirmtory.

Response: Thanks for your comments. In this study, we did not analyze the forms of Se present in plants. Instead, we examined how Se is transported in wheat after the application of exogenous selenite to the soil and in which parts of the plant it accumulates. Our ultimate goal is to increase Se accumulation in wheat grains to meet human daily intake requirements. Previous research has reported that selenite was more conducive to the accumulation of Se in grain than selenate. Therefore, selenite was selected for this study.

References:

Di, X.R.; Qin, X.; Zhao, L.J.; Liang, X.F.; Xu, Y.M.; Sun, Y.B.; Huang, Q.Q. Selenium distribution, translocation and speciation in wheat (Triticum aestivum L.) after foliar spraying selenite and selenate. Food Chem. 2023, 400, 134077.

No.1.6 In the conclusion, it is said"on the rhizosphere microenviron-460ment, growth, and antioxidant responses of wheat. ". The conclusion should reflect de described experimental design

Without a proper description of control pots, it is not appropriate to claim some conclusions such as “The results showed that the rhizosphere pH and dissolved organic carbon in the soil solution were higher than those of the non-rhizosphere soil solution, and the total Se and inorganic Se in the soil solu-463tion increased with the increase in Se application concentration” –

what was considered in the experiment as pots with and without rhizosphere?

What was the control pots to which the conclusions are drawn for Se contribution to DOC concentration? No where in the text, it is explained what DOC stands for.

Response: Thanks for your suggestions. In this study, the control experiment was an exogenous Se gradient of 0 mg Se kg^-1, meaning no exogenous sodium selenite was applied. When the application amount of exogenous Se was 0 mg kg^-1, the soil inside root bags was used to control for rhizosphere soil, and the soil outside root bags was used to control for non-rhizosphere soil. We added an explanation of DOC (dissolved organic carbon) in the materials and methods.

We appreciate the time and effort you put into our manuscripts. We sincerely hope that this revised manuscript has addressed all your comments and suggestions. We appreciated for your warm work earnestly, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Sincerely,

Corresponding author: Yuefeng Xu, Ph.D.

Address: College of Resources and Environment, Shanxi Agricultural University, Taigu, Shanxi 030801, China

E-mail: xuyf@sxau.edu.cn

Reviewer 2 Report

Comments and Suggestions for Authors

The article clearly and objectively addresses a current topic in Plant Physiology and Plant Mineral Nutrition, related to the effects of sodium selenite on the rhizosphere microenvironment, growth and antioxidant responses of wheat. In the Introduction, the authors cite classic scientific articles as well as more recent scientific articles, informing the reader about current knowledge related to the functions of selenium and food biofortification. There is also a good approach to the factors that control the availability of selenium in the soil.

Regarding the methodology, the authors detailed very well the procedures used, both in the implementation and conduct of the study, as well as in the enzymatic analyses. I have no suggestions or considerations for improving the conduct of the study and the evaluations performed. I believe, as previously stated, that the study was conducted very carefully.

I believe that the authors presented and discussed the results obtained very well. To this end, they used both basic and applied knowledge, cited in the references. In this regard, I also have no suggestions or considerations for improving the article.

In my opinion, the conclusion is very direct, consistent with the previous discussions and considerations.

Regarding the references, as I mentioned initially, classic scientific articles and also more recent scientific articles were cited, informing the reader of current knowledge related to the effects of sodium selenite on the rhizosphere microenvironment, growth and antioxidant responses of wheat. The tables and figures are very well presented, in a clear and didactic manner.

Author Response

Dear Reviewers,

The article clearly and objectively addresses a current topic in Plant Physiology and Plant Mineral Nutrition, related to the effects of sodium selenite on the rhizosphere microenvironment, growth and antioxidant responses of wheat. In the Introduction, the authors cite classic scientific articles as well as more recent scientific articles, informing the reader about current knowledge related to the functions of Se and food biofortification. There is also a good approach to the factors that control the availability of Se in the soil.

In my opinion, the conclusion is very direct, consistent with the previous discussions and considerations.

We appreciate the time and effort you put into our manuscript, as well as your approval of its content. We wish you every day happiness in life, every step in your career, and all the happiness and joy in your family. Finally, thank you once again for your approval of our manuscript.

Sincerely,

Corresponding author: Yuefeng Xu, Ph.D.

Address: College of Resources and Environment, Shanxi Agricultural University, Taigu, Shanxi 030801, China

E-mail: xuyf@sxau.edu.cn

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Please find my recommendation for the "Dynamic effects of sodium selenite on rhizospheric microenvironment, growth, and antioxidative responses of wheat (Triticum aestivum L.)" manuscript

L17: Replace "In" with "in"

L19-20: For clarity use only one decimals; Please consider that also in the whole body of manuscript where is possible

L43: The literature review of introduction section is little bit superficial, lacking depth and recent studies on selenium’s effects in wheat or similar crops. In my opinion this limits the context and justification for the research

L57-68: The manuscript must clearly identify the research gap. There are vaguely mentioned the need for investigation without specifying what is unknown or controversial in the field

L69-84: Please enlarge more with background information on selenium’s mechanisms, bioavailability, toxicity, and interactions with soil, those to ensure readers with a complete understanding of the topic

L116: Please explicitly describe the randomization process

L119: The authors mention "fifteen replications" but the total number of experimental units and their allocation is not specified. These need to be provided

L194: Please mention the data normality checks, homogeneity of variances, or post hoc tests

L201: In the "Results" section, the authors often states that differences are “significant” but does not specify exact p-values, confidence intervals, or effect sizes. This should be corrected

The statistical tests used are not consistently reported for each comparison, and there is no mention of corrections for multiple comparisons (such as Bonferroni correction), which is important given the number of parameters analyzed

L360: The discussion often restates findings without providing deeper mechanistic insights or connecting them to existing literature. For example, trends in soil pH and DOC are described, but there’s little explanation of the underlying processes or how these results compare to previous studies. This makes it difficult for readers to understand the broader significance of the findings

While changes in enzyme activities and selenium accumulation are reported, the manuscript does not explore the biological mechanisms behind these changes. There’s a missed opportunity to discuss how selenium affects plant metabolism or antioxidant pathways at the molecular level

Excessively speculative statements or statements presented as definitive without sufficient evidence should be avoided in the manuscript. For example, statements regarding the effectiveness of selenium in improving wheat growth are made without discussing possible confounding factors

In this section the limitations of the study, such as experimental design constraints, potential sources of variability, or external factors that could influence the results shoudl be stated and discussed also to ensure the research transparency and credibility

The authors should also consider the practical relevance of their findings. Eg., the discussion should address how selenium application might affect agricultural practices, food safety, or environmental management

Author Response

Dear Reviewers,

Please find my recommendation for the "Dynamic effects of sodium selenite on rhizospheric microenvironment, growth, and antioxidative responses of wheat (Triticum aestivum L.)" manuscript

No.1.1. L17: Replace "In" with "in".

Response: Thank you for pointing this out. We have made changes to it in the manuscript.

No.1.2. L19-20: For clarity use only one decimals; Please consider that also in the whole body of manuscript where is possible.

Response: We feel great thanks for your comments on our article. We agree that this is an important consideration. As you suggested, without prejudice to the presentation of the results, we have amended this question throughout the text.

No.1.3. The literature review of introduction section is little bit superficial, lacking depth and recent studies on selenium’s effects in wheat or similar crops. In my opinion this limits the context and justification for the research.

Response: Thank you for pointing this out. As you suggested, we have added recent findings on the effects of Se in wheat or similar crops to the Introduction. The details are as follows: “In crop production, Se fertilizer is often used to enhance plant Se content and improve resistance to abiotic stresses such as drought [11]. Previous studies have reported the beneficial effects of Se in plant growth and development, such as wheat, maize, rice, barley and quinoa [12-16]. Sadak and Bakhoum [16] demonstrated that, foliar spraying of Se, especially at a concentration of 50 mg L⁻¹, significantly enhanced the growth and productivity of quinoa plants under water stress. Gorni et al. [17] showed that Se improved growth parameters and yield of maize plants by promoting total carotenoids and antioxidant metabolism under water shortage stress. In another study, Se application (10 μmol L⁻¹) increased the concentration of chlorophyll and carotenoids in sugarcane leaves, and improved CO₂ assimilation rate, stomatal conductance, and internal CO₂ concentration [18]. Many studies have confirmed that low concentrations of Se are beneficial to plants. However, excessive levels of Se may be toxic to plants, leading to metabolic disorders, growth retardation, yellowing, necrosis, reduced protein biosynthesis, impaired function, and altered uptake and translocation of essential nutrients [19].”

References:

Ding, M.; Kong, Y.; Liu, J.; Li, H.; Li, S.; Yang, Y.; Zhang, C.; Xiao, C.; Rehman, M.; Maqbool, Z.; Fan, G.; Fahad, S.; Liu, K.; Zeng, C.; Deng, G. Evaluation of the Ameliorative Role of Soil Amendments and Selenium on Morphophysiological Traits, Oxidative Stress, and Quality Attributes of Wheat (Triticum aestivum L.) under Varying Drought Stress Conditions. J. Hazard. Mater. Adv. 2025, 18, 100693.

Yan, G.; Wu, L.; Hou, M.; Jia, S.; Jiang, L.; Zhang, D. Effects of Selenium Application on Wheat Yield and Grain Selenium Content: A Global Meta-Analysis. Field Crops Res. 2024, 307, 109266.

Peng, Z.; Sun, H.; Guo, Y.; Chen, Y.; Yin, X. Combining Depth and Rate of Selenium Fertilizer Basal Application to Improve Selenium Content and Yield in Sweet Maize. Agronomy 2025, 15, 775.

Geng, W.; Zhao, Y.; Mao, Z.; Wang X.; Wu N.;Xu X. The Effects of Combined Use of Black Soldier Fly Larvae Frass Fertilizer with Exogenous Selenium on Rice Growth and Accumulation of Heavy Metals. J. Soil Sci. Plant Nutr. 2022, 22, 5133–5143.

Cheng, C.; Li, Q.; Yi, Y.; Yang, H.; Coldea, T.E.; Zhao, H. Selenium Biofortification during Barley (Hordeum vulgare L.) Germination: Comparative Analysis of Selenate, Selenite, and Selenomethionine on Se-Protein Accumulation and Phenolic Acid Profile. Food Chem. 2025, 485, 144548.

Sadak, M.S.; Bakhoum, G.S. Selenium-Induced Modulations in Growth, Productivity, and Physiochemical Responses to Water Deficiency in Quinoa (Chenopodium quinoa) Grown in Sandy Soil. Biocatal. Agric. Biotechnol. 2022, 44, 102449.

Gorni, P.H.; Rodrigues, C.; Spera, K.D.; Correia, R.F.C.C.; Mendes, N.A.C.; Reis, A.R. Selenium Fertilization Enhances Carotenoid and Antioxidant Metabolism to Scavenge ROS and Increase Yield of Maize Plants Under Drought Stress. Plant Physiol. Biochem. 2025, 221, 109675.

Araujo, M.A.; Melo, A.A.R.; Silva, V.M.; Reis, A.R. Selenium Enhances ROS Scavenging Systems and Sugar Metabolism Increasing Growth of Sugarcane Plants. Plant Physiol. Biochem. 2023, 201, 107798.

Kamali-Andani, N.; Fallah, S.; Peralta-Videa, J.R.; Golkar, P.A. Comprehensive Study of Selenium and Cerium Oxide Nanoparticles on Mung Bean: Individual and Synergistic Effect on Photosynthesis Pigments, Antioxidants, and Dry Matter Accumulation. Sci. Total Environ. 2022, 830, 154837.

No.1.4. L57-68: The manuscript must clearly identify the research gap. There are vaguely mentioned the need for investigation without specifying what is unknown or controversial in the field.

Response: Thank you for your kind suggestion. As you suggested, we have identified research gaps. These are specified below: “Currently, the effects of exogenous Se application on the growth and Se content of wheat have been investigated [22-24], but the mechanism of transport and transformation of exogenous Se in the soil-wheat system after its application is not clear. Meanwhile, the interactions between root and rhizosphere soil play an important role in the effectiveness and mobility of soil nutrients.”

References:

Nawaz, F.; Ashraf, M.Y.; Ahmad, R.; Waraich, E.A.; Shabbir, R.N.; Bukhari, M.A. Supplemental Selenium Improves Wheat Grain Yield and Quality Through Alterations in Biochemical Processes Under Normal and Water Deficit Conditions. Food Chem. 2015, 175, 350–357.

Di, X.; Qin, X.; Zhao, L.; Liang, X.; Xu, Y.; Sun, Y.; Huang, Q. Selenium Distribution, Translocation and Speciation in Wheat (Triticum aestivum L.) After Foliar Spraying Selenite and Selenate. Food Chem. 2023, 400, 134077.

Yan, G.; Wu, L.; Hou, M.; Jia, S.; Jiang, L.; Zhang, D. Effects of Selenium Application on Wheat Yield and Grain Selenium Content: A Global Meta-Analysis. Field Crops Res. 2024, 307, 109266.

No.1.5. L69-84: Please enlarge more with background information on selenium’s mechanisms, bioavailability, toxicity, and interactions with soil, those to ensure readers with a complete understanding of the topic.

Response: We sincerely appreciate the valuable consideration. As you suggested, we have added these in the introductory section. The details are as follows:

Many studies have confirmed that low concentrations of Se are beneficial to plants. However, excessive levels of Se may be toxic to plants, leading to metabolic disorders, growth retardation, yellowing, necrosis, reduced protein biosynthesis, impaired function, and altered uptake and translocation of essential nutrients [19].

Liu et al. [28] found a significant increase of SOL-Se and EXC-Se content in the rhizosphere soil of L-selenocysteine-treated wheat. The study found that phosphate ions can displace Se(IV) from soil fixed site and enhance the concentration of Se in the soil solution, thereby improvng the bioavailability of Se[29]. In addition, Kikkert et al. [30] demonstrated that the presence of sulfate ions enhances the partitioning of Se in soil solution because sulfate competes with Se(VI) for the same adsorption sites on soil components.

References:

Liu, R.; Zhao, L.; Li, J.; Zhang, C.; Lyu, L.; Man, Y.B.; Wu, F. Influence of Exogenous Selenomethionine and Selenocystine on Uptake and Accumulation of Se in Winter Wheat (Triticum aestivum L. cv. Xinong 979). Environ. Sci. Pollut. Res. 2023, 30, 23887-23897.

Zhou, X.; Yang, J.; Kronzucker, H.J.; Shi, W. Selenium Biofortification and Interaction with Other Elements in Plants: A Review. Front. Plant Sci. 2020,11, 586421.

Kikkert, J.; Hale, B.; Berkelaar, E. Selenium Accumulation in Durum Wheat and Spring Canola as a Function of Amending Soils with Selenite, Selenate and/or Sulphate. Plant Soil 2013, 372, 629-641.

No.1.6. L116: Please explicitly describe the randomization process.

Response: We feel great thanks for your comments on our article. We have described them. The details are as follows:

All potted plants were randomized to ensure the accuracy of the test results and to eliminate subjective bias. A total of 75 pots were used for the experiment and were arranged in a 5x15 pattern. Each row and column was placed randomly, and grown under natural daylight conditions.

No.1.7. L119: The authors mention "fifteen replications" but the total number of experimental units and their allocation is not specified. These need to be provided.

Response: Thank you for your kind suggestion. We have added relevant content to the manuscript. They are detailed below:

To ensure the proper conduct of the experiment, each treatment was prepared with fifteen replications and each treatment selected four potted plants with uniform and good growth at 4, 6, and 8 weeks after planting.

No.1.8. L194: Please mention the data normality checks, homogeneity of variances, or post hoc tests.

Response: Thank you for pointing this out. We have added related information to the manuscript. They are detailed below:

The data were first tested for normality. The differences of biochemical parameters in soils and plants between the treatments were determined using one-way analysis of variance (ANOVA) based on homogeneity test and the Duncan's test.

No.1.9. L201: In the "Results" section, the authors often states that differences are “significant” but does not specify exact p-values, confidence intervals, or effect sizes. This should be corrected.

Response: Thank you for pointing this out. We have added (p<0.05) to the results wherever significant content exists.

No.1.10. The statistical tests used are not consistently reported for each comparison, and there is no mention of corrections for multiple comparisons (such as Bonferroni correction), which is important given the number of parameters analyzed.

Response: Thank you for pointing this out. Our data processing used only one-way ANOVA and did not involve multiple comparisons.

No.1.11. L360: The discussion often restates findings without providing deeper mechanistic insights or connecting them to existing literature. For example, trends in soil pH and DOC are described, but there’s little explanation of the underlying processes or how these results compare to previous studies. This makes it difficult for readers to understand the broader significance of the findings.

Response: Thanks for your suggestions. We have seen deficiencies in this aspect and have explained and added related information to the manuscript. The details are as follows:

The present study revealed that the pH and DOC content in the rhizosphere soil solution showed a downward parabolic trend as the concentration of added Se increased (Fig.1A-B). Hageman et al. [53] reported that plants increase rhizosphere soil pH when soil is acidic. Huang et al. [54] found that the pH of rhizosphere soil solution increased 0.10-1.50 units compared to nonrhizosphere soil solution when they investigated the effect of iron film on the rhizosphere Se content of rice root surface (pH = 5.09), which is consistent with the results of the present study. Yan et al. [55] obtained the same results in their study of cadmium adsorption by Cd-resistant microorganisms in the rhizosphere soil of rice, which may be related to the charge balance at the soil-root interface [56]. And root secretions and the root litter of plants are important sources of rhizosphere DOC [57]. The decline of DOC under high Se dose (10 mg kg⁻¹) may be because high concentrations of Se is toxic to wheat root system, which leads to a reduction in root activity and inhibition of root secretion. Furthermore, this may also be related to soil microbial activity. In high-Se soils, microbial respiration is intensified, which uses a large amount of DOM as an energy source and Se(VI) as an electron terminal receptor, thereby promoting the morphological transformation of Se and accelerating its binding to DOM [58].

References:

Hageman, S.P.; van der Weijden, R.D.; Weijma, J.; Buisman, C.J. Microbiological Selenate to Selenite Conversion for Selenium Removal. Water Res. 2013, 47, 2118–2128.

Huang, Q.Q.; Wang, Q.; Luo, Z.; Yu, Y.; Jiang, R.F. Effects of Root Iron Plaque on Selenite and Selenate Dynamics in Rhizosphere and Uptake by Rice (Oryza sativa). Plant Soil 2015, 388, 255-566.

Yan, Z.X.; Li, Y.; Peng, S.Y.; Wei, L.; Zhang, B.; Deng, X.Y.; Zhong, M.; Cheng, X. Cadmium Biosorption and Mechanism Investigation Using Two Cadmium-Tolerant Microorganisms Isolated from Rhizosphere Soil of Rice. J. Hazard. Mater. 2024, 470.

Bertin, C.; Yang, X.; Weston, L.A. The Role of Root Exudates and Allelochemicals in the Rhizosphere. Plant Soil 2003, 256, 67–83.

Bravin, M.N.; Martí, A.L.; Clairotte, M.; Hinsinger, P. Rhizosphere Alkalisation—A Major Driver of Copper Bioavailability Over a Broad pH Range in an Acidic, Copper-Contaminated Soil. Plant Soil 2009, 318, 257–268.

Wang, J.L.; Liu, X.M.; Li, W.J.; Tu, Y.L.; Deng, H.; Du, J.W. Deciphering Heavy Metal Adsorption Capacity of Soil Based on Its Physicochemical Properties and Adsorption Reaction Time Using Machine Learning. J. Environ. Chem. Eng. 2025, 13, 116913.

No.1.12. While changes in enzyme activities and selenium accumulation are reported, the manuscript does not explore the biological mechanisms behind these changes. There’s a missed opportunity to discuss how selenium affects plant metabolism or antioxidant pathways at the molecular level.

Response: Thank you for your kind suggestion. We have added related information to this section. The details are as follows:

For instance, moderate amounts of Se have been shown to attenuate oxidative stress under cadmium contamination environment, as evidenced by reductions in O₂^-, H₂O₂, and malondialdehyde in rice tissues[75], and to modulate reactive oxygen metabolism[76].

References:

Qu, L.; Xu, J.; Dai, Z.; Elyamine, A.M.; Huang, W.; Han, D.; Jia, W. Selenium in Soil-Plant System: Transport, Detoxification and Bioremediation. J. Hazard. Mater. 2023, 452, 131272.

Huang, G.X.; Ding, C.F.; Li, Y.S.; Zhang, T.L.; Wang, X.X. Selenium Enhances Iron Plaque Formation by Elevating the Radial Oxygen Loss of Roots to Reduce Cadmium Accumulation in Rice (Oryza sativa L.). J. Hazard. Mater. 2020, 398, 122860.

No.1.13. Excessively speculative statements or statements presented as definitive without sufficient evidence should be avoided in the manuscript. For example, statements regarding the effectiveness of selenium in improving wheat growth are made without discussing possible confounding factors.

Response: Thank you for your kind suggestion. We have added related information to this section. The details are as follows:

Moderate application of Se activates nitrate reductase and glutamine synthetase, which can stimulate nitrogen metabolism[60]. This stimulation favors an increase in nitrogen levels, which in turn favors plant development and growth, as well as the photosynthesis process and sugar metabolism [61]. Selenium competes for adsorption sites with other anions such as phosphate and sulfate, so Se application may increase phosphorus and sulfur levels and thus promote plant growth, a possibility that needs further verification [62,63].

References:

Shahid, M.A.; Balal, R.M.; Khan, N.; Zotarelli, L.; Garcia-Sanchez, F. Selenium Impedes Cadmium and Arsenic Toxicity in Potato by Modulating Carbohydrate and Nitrogen Metabolism. Ecotoxicology Environ. Saf. 2019, 180.

Cipriano, P.E.; Júnior, M.S.; Souza, R.R.D.; Silva, D.F.D.; Silva, R.F.D.; Silva, M.L.D.S.; Faquin, V.; Guilherme, L.R.G. Selenium Inorganic Sources Applied to Soil: Effects on Gas Exchange and Anatomical Changes of Radishes. S. Afr. J. Bot. 2024,170, 17.

Mendes Araujo, A.; Lima Lessa, J.H.D.; Chanavat Gustavo, L.; Curi, N.; Lopes, G. How Sulfate Content and Soil Depth Affect the Adsorption/Desorption of Selenate and Selenite in Tropical Soils? Rev. Bras. Cienc. Solo. 2020, 44, e0200084.

Vila, P.A.; Faquin, V.; Vila, F.W.; Kachinski, W.D.; Carvalho, G.S.; Guilherme, L.R.G. Phosphorus and Sulfur in a Tropical Soil and Their Effects on Growth and Selenium Accumulation in Leucaena leucocephala (Lam.) de Wit. Environ. Sci. Pollut. Res. 2020, 27, 44060-44072.

No.1.14. In this section the limitations of the study, such as experimental design constraints, potential sources of variability, or external factors that could influence the results shoudl be stated and discussed also to ensure the research transparency and credibility.

Response: Thank you for your kind suggestion. We think your suggestion is very good and we have added related information to this section. The details are as follows:

The data presented herein were derived from pot experiments; however, numerous conditions could not be compared with those in field conditions, such as light, temperature, and humidity. Secondly, the soil type and nutrient status in the field environment cannot be fully reflected, and the root growth of the plants may be limited. Therefore, it is possible for the data to differ from the field data. However, the study did not include measurements of the changes in microorganisms in the soil after Se treatment. Consequently, the effects of Se treatment on soil microorganisms cannot be interpreted at the microbial level. Consequently, a field experiment will be conducted to augment the study of soil microorganisms in a subsequent study.

No.1.15. The authors should also consider the practical relevance of their findings. Eg., the discussion should address how selenium application might affect agricultural practices, food safety, or environmental management.

Response: Thank you for your kind suggestion. We have added this section as follows:

In recent decades, with the growing demand for Se, various Se-enriched products have emerged, such as Se-enriched tea, Se-enriched rice, and Se-enriched millet. The application of Se in agriculture has also become increasingly widespread. Although Se is important, its bioavailability in soil is typically low, which limits its effectiveness in enhancing crop yields and nutritional quality[82]. Se that remains in the soil after not being absorbed by plants may contaminate soil and water bodies. Zhai et al. [83] studied soil leaching and found that when the exogenous Se concentration was 1, 3, and 6 mg kg⁻¹, the Se content in the soil leachate was 0.06–0.24, 0.25–0.84, and 0.60–1.65 mg L⁻¹, respectively.exceeding the standard limit of the Chinese groundwater environmental quality standards (< 0.01 mg L⁻¹) (GB/T 14848-2017), indicating that the leaching risk of Se in Se-amended soils is relatively high and poses certain environmental risks.

References:

Alshaal, T.; Szabolcsy, É.D.; Fári, M.; Veres, S.; Kaszás, L.; Kovács, Z.; Eissa, F.; Elhawat, N. Agricultural Sustainability and the Challenges of Selenium Nanoparticles (SeNPs): Their Role in Supporting the Environmental Economy. Plant Stress 2025, 16, 100846.

Zhai, H.; Xue, M.; Du, Z.; et al. Leaching Behaviors and Chemical Fraction Distribution of Exogenous Selenium in Three Agricultural Soils Through Simulated Rainfall. Ecotoxicology Environ. Saf. 2019, 173, 393-400.

Once again, we acknowledge your comments very much, which are valuable in improving the quality of our manuscript. We hope that the changes we have made resolve all your concerns about the article. Thank you very much for your help.

Sincerely,

Corresponding author: Yuefeng Xu, Ph.D.

Address: College of Resources and Environment, Shanxi Agricultural University, Taigu, Shanxi 030801, China

E-mail: xuyf@sxau.edu.cn

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript discusses the dynamic effects of sodium selenite on rhizospheric microenvironment, growth, and antioxidative responses of wheat (Triticum aestivum L.).

The language is fluent. All answers should be included in the revised text.

Some points are raised below.

72-74: The criterion for the various categories of Se is not the same.
78-80: unclear.
83: Perhaps delete “therefore”?
109: Why use deionized water? Se would not be present in tap water; besides ions present in water are useful for plant growth. Please, explain.
123-4: unclear.
189: Absorbance or λmax? Please, explain.
190: Explain “Ug-1FW”.
227: How did the authors measure “inorganic Se”? Explanation is needed and a new passage in the experimental section.
243-5: Please, explain why.
291: Table 1?
292-4: unclear.
368-70: unclear.
423-5: Please, explain why.
430-6: unclear. Further explanation is needed.

Author Response

Dear Reviewers,

The manuscript discusses the dynamic effects of sodium selenite on rhizospheric microenvironment, growth, and antioxidative responses of wheat (Triticum aestivum L.). The language is fluent. All answers should be included in the revised text. Some points are raised below.

No.1.1 72-74: The criterion for the various categories of Se is not the same.

Response: We are very grateful for your advice. In the study of Se, soil Se is categorized according to valence states as -2, 0, +4, +6. And according to the water solubility and binding strength, Se is categorized into soluble Se (SOL-Se), exchangeable Se (EXC-Se), ferromanganese oxide-bound Se (FMO-Se), organic matter-bound Se (OM-Se), and residual Se (RES-Se). It is the latter that we have used.

No.1.2 78-80: unclear.

Response: We are very grateful for your advice. We have added the related content in the revised manuscript as follows:

The study found that phosphate ions can displace Se(IV) from soil fixed site and enhance the concentration of Se in the soil solution, thereby improvng the bioavailability of Se [29]. In addition, Kikkert et al. [30] demonstrated that the presence of sulfate ions enhances the partitioning of Se in soil solution because sulfate competes with Se(VI) for the same adsorption sites on soil components.

Matos et al.[38] found that the negative charge on soil particle surfaces typically increases with rising pH, leading to enhanced electrostatic repulsion between selenate and selenite ions and the negative charges, thereby promoting the release of Se from the soil solid phase and increasing its bioavailability. Wang et al. [39] found that soil Se can be adsorbed, chelated, or complexed by organic compounds, thereby reducing the amount of available Se in the soil.

References:

Matos, R.P.; Lima, V.M.P.; Windmöller, C.C.; Nascentes, C.C. Correlation Between the Natural Levels of Selenium and Soil Physicochemical Characteristics From the Jequitinhonha Valley (MG), Brazil. J. Geochem. Explor. 2017, 172, 195-202.

Wang, D.; Xue, M.Y.; Wang, Y.K.; Zhou, D.Z.; Tang, L.; Cao, S.Y.; Wei, Y.H.; Yang, C.; Liang, D.L. Effects of Straw Amendment on Selenium Aging in Soils: Mechanism and Influential Factors. Sci. Total Environ. 2019, 657, 871-881.

No.1.3 83: Perhaps delete “therefore”?

Response: We are very grateful for your advice. We have deleted it in the revised manuscript.

No.1.4 109: Why use deionized water? Se would not be present in tap water; besides ions present in water are useful for plant growth. Please, explain.

Response: We are very grateful for your advice. The purpose of using deionized water is to eliminate possible interference with Se effectiveness caused by other factors during the dissolution of Se.

No.1.5 123-4: unclear

Response: We are very grateful for your advice. As you said, we have modified it as follows: "To ensure the proper conduct of the experiment, each treatment was prepared with fifteen replications and each treatment selected four potted plants with uniform and good growth at 4, 6, and 8 weeks after planting."

No.1.6: 189: Absorbance or λmax? Please, explain.

Response: We are very grateful for your advice. The SOD measurement is strictly in accordance with the kit instructions. The reason for choosing absorbance is that through the xanthine and xanthine oxidase reaction system produces superoxide anion (O^2-), then nitrogen blue tetrazolium can be reduced by O^2-to blue dirty, the latter having absorption in the 560 nm.

No.1.7: 190: Explain “U g^-1 FW”.

Response: We are very grateful for your advice. The unit of U g^-1 FW is expressed as units of enzyme activity per gram of fresh weight tissue.

No.1.8 227: How did the authors measure “inorganic Se”? Explanation is needed and a new passage in the experimental section.

Response: We are very grateful for your advice. We describe the determination of inorganic Se in “2.4.1”, as shown in red:

To evaluate Se speciation and concentration in the soil solution, we used 0.01 M CaCl₂ solution, which is often used to assess the bioavailability of plant nutrients in soils[42,43]. Twenty grams of soil was mixed with 50 mL of 0.01 M CaCl₂ solution in 100-mL centrifuge tubes and shaken for 2 h at 20 ℃ in darkness on a reciprocal shaker. The mixture was then centrifuged at 3000 rpm for 25 min, and the supernatant was filtered over 0.45 µm membrane.

The extracted soil solutions were separated into several sub-samples. The pH and dissolved organic carbon (DOC) were determined using a pH meter (PHB-4) and TOC analyzer (Shimadzu, Japan) respectively. The detailed procedure for soil Se speciation analysis in this study was described by Kulp and Pratt [44]. Inorganic Se concentration was measured by heating the extract for 30 min at 100 ℃ in 6 M HCl solution. Total Se concentration was determined by adding 2 mL HNO₃/70% HClO₄ (1:1) mixture to 5 ml extract, heating the solution for 30 min at 170 °C, and then for 30 min at 100 °C in 6 M HCl solution.

No.1.9 243-5: Please, explain why.

Response: We are very grateful for your advice. The possible reason for the initial decrease is that the non-rhizosphere soil lacks a continuous input of root secretions, and Se may be re-adsorbed by soil minerals or converted into insoluble forms. The possible reason for the increase in the later stage is that as wheat grows, root aging or litter increases, and soil organic matter decomposition may release fixed Se. Under the low Se content (0-1 mg kg^-1 treatment), the influence of the above factors is more obvious.

No.1.10 291: Table 1?

Response: We are very grateful for your advice. We did find this problem here, and we have correctly changed it to "Table 3".

No.1.11 292-4: unclear.

Response: We are very grateful for your advice. As described in the discussion, the cause of this phenomenon may be due to the toxicity of high Se concentrations to the roots, which in turn continue to transport Se to the shoot.

No.1.12 368-70: unclear.

Response: We are very grateful for your advice. We have redescribed it as "Wang et al. [46] found that the proportion of effective Se to total Se was not more than 10% at different Se levels (0.07-1.23 mg kg⁻¹)."

No.1.13 423-5: Please, explain why.

Response: We are very grateful for your advice. We explain this in the previous paragraph of this section as well. It is as follows:

Selenite is mainly taken up by plants through silico transporter proteins and also possibly the metabolism-dependent active process mechanisms via phosphate transporter proteins, resulting in its accumulation in the root system with minimal translocation to the shoot [63]. Zhang [64] also came to a similar conclusion, i.e., selenite is poorly mobile and usually stays in the roots and converted to other forms.

No.1.14 430-6: Unclear. Further explanation is needed.

Response: We are very grateful for your advice. We have rewritten it. The details are as follows:

This is in agreement with the findings of Fu [65], who reported that sodium selenite treatment resulted in higher accumulation capacity in wheat seeds and roots than sodium selenate treatment, leading to a TF value less than 1 for sodium selenite treatment.

Sincerely,

Corresponding author: Yuefeng Xu, Ph.D.

Address: College of Resources and Environment, Shanxi Agricultural University, Taigu, Shanxi 030801, China

E-mail: xuyf@sxau.edu.cn

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Manuscript Agronomy -3643415-peer-review v2, entitled

Dynamic effects of sodium selenite on the rhizospheric microenvi-2ronment, growth, and antioxidative responses of wheat (Triti-3cum aestivum L.) by Fang Qin et al addresses a relevant topic and may be of interest to readers and peers in the field. In their revised 2 manuscript, the authors have improved their method description to make it more clear. However, this does not address the original flaw in the conceptual experiment design of a single experiment conducted in May 2019. Replicating each treatment 4X in a single experiment does not warrant reproducibility of the data. To be convincing, with supportive conclusion, at least repeating the expecting the experiment at least twice in time or space of the same treatments are required Whereas the data collection and acquisitions appear to be well conducted, the experimental design from which the data were collected is flawed for ensuring reproducibility of the data. The reported and discussed data appeared to be only confirmatory and their originality not pointed out. As a second review, I would recommend a final rejection of this manuscript, and recommend to the authors to carry a second experiments to collect growth, roots soil parameters to confirm the current data for drawing strong conclusions and be complement by their current biochemical data.

Specific concerns can be found below.

Major concerns:

Abstract

First sentence of abstract not clear

Introduction

Literature review has been improved
English quality can be improved throughout the text

Methodology

Use of Rhizobag now better explained
Data generated from a single experiment although 4 replications/unit were reported – the flaw in the conceptual design remains and not-addressed.

Result section

Data on the figures are well presented, but their contents and validity are contingent to the design they derive from. – How repeatable the experiments can be is not addressed.

Discussion

Clarity of discussion has improved
Authors fail to highlight is the originality of their finding. As presented and discussed the data appear to be purely confirmatory.
The statement on lines 496-497 seems not accurate and lacks rationality. If this reviewer follows the author:
How does Se move from roots to seed then if usually stays in the root? and
Do all Se applied through foliar application stay in the leave if not mobile?
The discussions should be improved.

Conclusions.

Major concerns remain (1) the reproducibility and (2) the originality of the data which are not clear at this point with the presented design, data, and discussions

Comments on the Quality of English Language

The Manuscript Agronomy -3643415-peer-review v2, entitled

Specific concerns can be found below.

Major concerns:

Abstract

First sentence of abstract not clear

Introduction

Literature review has been improved
English quality can be improved throughout the text

Methodology

Use of Rhizobag now better explained
Data generated from a single experiment although 4 replications/unit were reported – the flaw in the conceptual design remains and not-addressed.

Result section

Data on the figures are well presented, but their contents and validity are contingent to the design they derive from. – How repeatable the experiments can be is not addressed.

Discussion

Clarity of discussion has improved
Authors fail to highlight is the originality of their finding. As presented and discussed the data appear to be purely confirmatory.
The statement on lines 496-497 seems not accurate and lacks rationality. If this reviewer follows the author:
How does Se move from roots to seed then if usually stays in the root? and
Do all Se applied through foliar application stay in the leave if not mobile?

The discussions should be improved.

Conclusions.

Major concerns remain (1) the reproducibility and (2) the originality of the data which are not clear at this point with the presented design, data, and discussions

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you very much for considering my recommendations in your manuscript. Reading carefully the manuscript I noticed several things that in my opinion should be clarified by authors. Please se below:

In results section the authors mention “significant” differences and use a p < 0.05 threshold, but do not provide detailed statistical test results (such as F-values, degrees of freedom, or exact p-values). Please improve these
Data are expressed as mean ± SD, but it’s not clear if assumptions of normality and homogeneity of variance were tested before using ANOVA. Please clarify
Broad statements as "increased" should be followed by numerical magnitude of increase and/or variability, and statistical significance

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

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Dynamic Effects of Sodium Selenite on the Rhizospheric Microenvironment, Growth, and Antioxidative Responses of Wheat (Triticum aestivum L.)

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