Organic Acids Metabolic Response and Transcription Factor Expression Changes of Highland Barley Seedlings Under Na₂SeO₃ Treatment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall is an acceptable manuscript but to much data was placed on the figure, some could go to supplementary material. A model on the effect of selenium from all this data would be welcome as it would summarize better all the information that has been generates. With regard to the figures, the authors use several different formats to show graphs, they should homogenize to one style.
Figure 1, 1a is hard to see to small and there is a lack of definition of t1, ck and t2 in the figure legend
Figure 3, 3d needs to use different colors as not to compare with A to C from this figure as the data is very different. Error bars are not seen
Figure 4 C is too small to see anything of use on the dendrogram

Comments on the Quality of English Language

none

Author Response

Dear editor,

 

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft, the detailed corrections are listed below.

 

 

Q1. Overall is an acceptable manuscript but to much data was placed on the figure, some could go to supplementary material.

R1# The figure 5D was changed supplementary Figure S2.

The figure 7A was changed supplementary Table S5.

 

Q2. A model on the effect of selenium from all this data would be welcome as it would summarize better all the information that has been generates.

R2# We checked the whole manuscript and summarized the information. In lines 276-278, lines 299-302, lines 336-339, lines 366-368. The rest of the content is in the article, and we did not repeat it

 

Q3. With regard to the figures, the authors use several different formats to show graphs, they should homogenize to one style.
R3.# The figure 3 was changed and same to the style of figure 1, 2.

 

Q4. Figure 1, 1a is hard to see to small and there is a lack of definition of t1, ck and t2

R4# The figure 1A was enlarged. The definition of t1, ck and t2 was added.

 

Q5. In the figure legend Figure 3, 3d needs to use different colors as not to compare with A to C from this figure as the data is very different.

R5# Colours in the figure 3D was changed and deleted the comparison between A to C in lines 340.

 

Q6. Error bars are not seen Figure 4 C is too small to see anything of use on the dendrogram.

R6# Four repetitions of metabolites were used in figure 4C, and there were not error bars. A total of 759 metabolites were identified for cluster analysis.  

We added “In metabolomics analysis, the contents of or so two-thirds of metabolites increased and one-third of metabolites decreased under Se2 treatment.”

In lines 366-368.

 

We tried our best to improve the manuscript and made some changes marked in red in revised paper which will not influence the content and framework of the paper. We appreciate for editors and reviewers' warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

-Please clarify why you use Na2SeO3 as a Se source, since there is a high potential risk of Na toxicity.

- mention Se toxicity 

-mention Na toxicity 

-Mention the daily dose of Se that the human body needs 

-Uptake and transport of Se by the plants

-Se, it may be beneficial for plants under stress, not under normal conditions 

-L166, there is no method named "thiobarbituric method" for soluble sugars, check and correct

-L196, how do you prepare the samples (50mg), dry, fresh, lyophilization.....?

-Provide a clear link between all the parameters during the discussion section 

-Other parameters, such as the accumulation factor plant/soil, will be useful

- What is the fate of the remaining Se in the soil? How much Se stay? What is the initial concentration before the Se application? 

- correct axis title fig 3A, B, and C

-The human body needs around 55 micrograms per day. According to your results, the plant accumulates more than 10 mg/kg in the shoot part. Is there any risk of toxicity after consumption of this plant 

- Many sentences are repeated many times, like L319 ...... was replicated four times ..., you can start the results analysis directly without starting with an introduction sentence 

-L462 and L463 should be in the materials and methods section, not the results section

-Why does the expression of some genes decrease? Is that a sign of Se toxicity?

- Can you explain why you use correlation analysis everywhere

- L566 and L567, what is the link between MDA and inhibition of antioxidant activities?

-High Proline content under T2, maybe a sign of Se toxicity and osmotic stress

-How Se+ can act as a pro-oxidant 

-L587 and L588,  Why Se under T2 inhibit SOD POD APX

-Did you assess and check the impact of Na release from NaSeO?

What are your recommendations regarding the use of selenium: under what conditions and in what form should we apply it, knowing that the plant uses it in very low concentrations, do we need to apply an exogenous Se? 

Author Response

Dear editor,

 

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft, the detailed corrections are listed below.

 

Q1 -Please clarify why you use Na₂SeO₃ as a Se source, since there is a high potential risk of Na toxicity.

(1)- mention Se toxicity 

(2)-mention Na toxicity 

(3)-Mention the daily dose of Se that the human body needs 

(4)-Uptake and transport of Se by the plants

(5)-Se, it may be beneficial for plants under stress, not under normal conditions 

R1#After dissolution, Na₂SeO₃ can stably release selenium ions, which facilitates precise control of selenium concentration and is in line with conventional research methods in the same field.

(1) Se toxicity was added in lines 65-70.

In plants, superfluous Se interferes with nutrient absorption, inhibits the formation of photosynthetic pigments, causes oxide damage, and induces genotoxic effects. Additionally, elevated Se levels above this threshold impede plant growth and development, thus reducing grain yield. High selenium concentrations can cause scorch-like damage to leaves, hinder branch development, reduce root growth, and even result in plant death.

(2) The concentration of Na₂SeO₃ used in this study was 0.01 g/kg~0.2 g/kg, in which the sodium toxicity is much lower than Se toxicity in most plants. Therefore, the effect of Na toxicity on plant growth can be ignored. Under the treatment of sodium selenite, the toxic effects of selenium are mainly studied, which have been reported in many literature. [Wang Q, Hu J, Hu H, Li Y, Xiang M, Wang D. Integrated eco-physiological, biochemical, and molecular biological analyses of selenium fortification mechanism in alfalfa. Planta. 2022 Nov 12;256(6):114. doi: 10.1007/s00425-022-04027-6. PMID: 36370252.]

0.2g/kg of sodium selenite is equivalent to 0.001156mol/L, and the molar concentration of sodium ions is 0.002312mol/L, which is equivalent to 2.312mmol/L. The toxic effect of sodium ions is very small. There are literature reports that the toxic concentration of sodium ions is at least 100mol/L or higher, therefore, in this article, the main focus is on selenium toxicity, without studying sodium toxicity. If higher concentrations of sodium selenite are involved in subsequent research, a separate sodium ion control will be considered to distinguish the effects of the two.

(3)We added the se that the human body needs “According to World Health Organization (WHO) standards, the recommended dose of selelium for adults is 55 μg/d, while the maximum tolerable adult intake without side effects is set at 400 μg/d. Cereal crops serve as the primary source of dietary selelium for humans. Nonetheless, their selelium content is typically low, ranging from 0.01 to 0.55 μg/g in cereals.”In lines 111-115

(4)We added the uptake and transport of Se by the plants in lines 51-54.

(5)Se may be beneficial for plants in lines 55-64.

 

Q2  -L166, there is no method named "thiobarbituric method" for soluble sugars, check and correct

R2# We added the The soluble sugar content measurement in lines 182-183.

 The soluble sugar content was determined using the anthrone method.

 

Q3  -L196, how do you prepare the samples (50mg), dry, fresh, lyophilization.....?

R3# We described the preparation in lines 217.

We used 50 mg lyophilization sample

 

Q4  -Provide a clear link between all the parameters during the discussion section 

R4# We modified the link between all the parameters in discussion section.

 

Q5  -Other parameters, such as the accumulation factor plant/soil, will be useful

R5# The calculation formula for Se accumulation factor is the total selenium content of the measured plant or soil divided by the total Se content applied to the soil. Under Se1 treatment, Se accumulation factor in soil, or plant was 0.0283, or 0.2057, respectively. Under Se2 treatment, Se accumulation factor  in soil or plant was 0.0276, or 0.2149, respectively. There was no significant change in the Se accumulation factor of plants or soil.

In lines 332-335.

 

Q6 - What is the fate of the remaining Se in the soil? How much Se stay? What is the initial concentration before the Se application? 

R5# Partial selenium is converted by microorganisms.

In theory, the selenium content obtained after four treatments with T1 concentration is 0.0576 g/kg in soil, and the selenium content obtained after four treatments with T2 concentration is 0.576 g/kg in soil. The remaining selenium is the selenium content we measured.

We measured the total selenium content in the soil control group to be 0.649mg/kg before Se treatment.

 

Q7 - correct axis title fig 3A, B, and C

R7#We corrected axis title in fig 3A, B, and C

 

Q8 -The human body needs around 55 micrograms per day. According to your results, the plant accumulates more than 10 mg/kg in the shoot part. Is there any risk of toxicity after consumption of this plant 

R8# This study focuses on the effects of selenium concentration on the physiological response of barley seedlings (such as growth indicators, antioxidant enzyme activity, etc.), rather than food safety assessment. The main edible part of barley is mature grains, and young seedlings are usually not used as human food. The accumulation of selenium in their stems does not directly reflect food security risks. If it involves the use of feed, further evaluation is needed, but no relevant experiments were conducted in this study.

 

Q9 - Many sentences are repeated many times, like L319 ...... was replicated four times ..., you can start the results analysis directly without starting with an introduction sentence 

R9# I have deleted the repeated statements and analyzed the results directly.

 

Q10 - L462 and L463 should be in the materials and methods section, not the results section

R10# I have added the content of L462 and L463 to the materials and methods section in lines 248-249.

 

Q11  -Why does the expression of some genes decrease? Is that a sign of Se toxicity?

R11# Yes, Reduced gene expression is one of the potential signs of selenium poisoning.

 

Q12- Can you explain why you use correlation analysis everywhere

R12# Through correlation analysis, we can explore the correlation between variables,

reduce data dimensions and simplify analysis.

In our study, we investigated the correlation between gene expression levels and organic acid metabolites, revealing potential regulatory mechanisms. Provide clues for further in-depth research.

 

Q13 - L566 and L567, what is the link between MDA and inhibition of antioxidant activities?

R13# MDA is a hallmark product of lipid peroxidation. With the increase of selenium concentration, the MDA content significantly increased, and the degree of lipid oxidation on the cell membrane increased. Under Se2 treatment, the activity of antioxidant enzymes decreased, which led to a decrease in cellular antioxidant capacity.

 

Q14 - High Proline content under T2, maybe a sign of Se toxicity and osmotic stress

R14# Yes, proline plays a dual role in plants' response to abiotic stress: it is both a cytoplasmic osmotic regulator and an active oxygen scavenger. Excessive selenium can trigger proline accumulation by inducing oxidative stress and ion imbalance. In this study, the selenium content in plant leaves of the Se2 treatment group was significantly higher than that of the control group, indicating that selenium accumulation in the body has reached a potential toxic level. Excessive intake of selenium may interfere with enzyme activity, disrupt cell membrane structure, and induce proline as a stress protective substance. Therefore, the association between elevated proline levels under Se2 treatment and selenium toxicity or osmotic stress is consistent with existing theories.

 

Q15 - How Se+ can act as a pro-oxidant 

R15# Exceeding a certain concentration causes cellular antioxidant activity, high concentration direct damage: When the free selenium ions are too high, they directly attack lipids, proteins, and DNA, causing lipid peroxidation, thiol oxidation, and DNA damage.

 

 

Q16 - L587 and L588,  Why Se under T2 inhibit SOD POD APX

R16# The reduction of enzyme activity by high selenium treatment is a synergistic effect of multiple mechanisms

1. High concentration selenium treatment directly destroys enzyme structure and reduces enzyme protein activity through metal ion competition, thiol modification, and other methods
2. Selenium induces ROS overload, causing oxidative damage to enzyme proteins and inhibiting enzyme gene expression, thereby reducing enzyme activity.

 

Q17  -Did you assess and check the impact of Na release from NaSeO?

R17# This study used sodium selenite (Na₂SeO₃) as the selenium source and did not specifically evaluate the independent effect of Na ⁺ release, which is a limitation of the study design. In studies similar to plant selenium stress, when using Na₂SeO₃ or K₂SeO₃ as selenium sources, it is usually assumed that the Na ⁺/K ⁺ concentration is low and its effects are not evaluated separately.

 

Q18  What are your recommendations regarding the use of selenium: under what conditions and in what form should we apply it, knowing that the plant uses it in very low concentrations, do we need to apply an exogenous Se? 

R18# Attention should be paid to the sensitivity differences of plants to selenium during application, and the dosage should be strictly controlled for sensitive crops such as grasses. From the perspective of selenium application forms, inorganic selenium has low cost and fast absorption, making it suitable as a soil based fertilizer; Organic selenium has low toxicity and is more suitable for foliar spraying on edible crops; Nano selenium has high utilization efficiency and is suitable for high value-added crops. Whether to apply exogenous selenium needs to be judged based on soil conditions, crop types, and production goals. When dealing with stress such as heavy metal pollution and drought, the application of exogenous selenium can significantly improve plant growth and quality; If the soil has sufficient selenium content or non food crops are planted, there is usually no need for additional application. In areas with selenium deficiency in soil, plants are prone to selenium deficiency and require external supplementation. In terms of application method, foliar spraying has strong targeting and high utilization rate, suitable for selenium supplementation in the later stage of growth. Soil application as a base fertilizer can meet the needs of crops throughout their entire growth period. Seed treatment is beneficial for enhancing stress resistance during the seedling stage. Regardless of the method, the principle of "low-dose precision" should be followed to avoid toxicity risks and environmental pollution, in order to achieve a balance between the efficiency and safety of selenium application.

We tried our best to improve the manuscript and made some changes marked in red in revised paper which will not influence the content and framework of the paper. We appreciate for editors and reviewers' warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors employed transcriptomic and metabolomic approaches to understand the metabolism of highland barley under selenium treatment. By assessing two Se concentration, the authors concluded 0.02 g/Kg provided the best results on plant growth. In contrast, higher concentration decreased photosynthesis. In contrast, the high selenium concentration T2 (0.2 g/kg) significantly decreased photosynthesis. It is still necessary to understand the mecanisms to vinculate the impressive family of TF turned on with the metabolites found.

Several requests are located alongside the text within the pdf file. Remember to write scientific names in italics. Go forward with your research.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

To improve the understanding of the present contribution, English quality could be reviewed. 

Author Response

Dear editor,

 

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft, the detailed corrections are listed below.

 

The authors employed transcriptomic and metabolomic approaches to understand the metabolism of highland barley under selenium treatment.

Q: By assessing two Se concentration, the authors concluded 0.02 g/Kg provided the best results on plant growth. In contrast, higher concentration decreased photosynthesis. In contrast, the high selenium concentration T2 (0.2 g/kg) significantly decreased photosynthesis. It is still necessary to understand the mecanisms to vinculate the impressive family of TF turned on with the metabolites found.

R# We fully agree with the reviewer's suggestion that mechanism research is crucial for understanding the pathway of selenium. However, this study only detected macroscopic indicators of photosynthesis and did not synchronously measure key enzyme activity data. The phenotype observed under high selenium treatment may be driven by multiple factors, and the interpretation of a single mechanism requires more experimental evidence to support. But it provides important concentration references and phenotype clues for subsequent research. Thank you for the constructive comments provided by the reviewer. Exploring relevant mechanisms will be one of our future research priorities.

Several requests are located alongside the text within the pdf file. Remember to write scientific names in italics. Go forward with your research.

Q1 Pleasese consider change T1 and T2 to low and high selenimu concentration, as T is employed to describe filial generations.

R1# I have changed T1 (0.02g/kg) to Se1 (0.02g/kg), T2 (0.2g/kg) to Se2 (0.2g/kg), and made corresponding modifications to the images and text throughout the text.

 

Q2 Sentence deletion, word replacement, and font italicization issues

R2# “essential trace nutrien” changed to “essential trace mineral” in lines 46.

Deleted “not only increases the selenium content in plants but also” in lines 55-56.

“aloe vera and Hordeum“changed to “Aloe vera and Hordeum” in lines 63 and 104.

resistance changed to tolerance in lines 107.

Ultrapure changed to distilled in lines 151

level changed to concentrations in lines 407

 

Q3 This section in material and methods needs to be rewritten. Describe the methods in the way they were performed (in past). Do not use the present tense(2.2 and 2.5)

R3# I have rewritten the materials and methods section using the past tense. Checked and modified the tense of the entire text.

 

Q4 “Compared to the CK, the contents of inorganic selenium, organic selenium, and total selenium in the soil under T1 treatments increased.”therefore thre was not difference. Rephrase please

R4# Compared to the CK, the contents of inorganic selenium, organic selenium, and total selenium in the soil under Se1 treatments increased, and did not reach a statistically significant level (Figure 3A). In lines 311-313.

 

Q5 please provide the amounts, instead of the times the Se increased

R5# I have provide the amounts as follows:

In contrast, under Se2 treatment, the inorganic selenium and total selenium content in the soil increased from 0.51mk/kg and 0.649mg/kg to 12.83mk/kg and 15.941mg/kg, respectively (p<0.05, Figure 3A).

Compared to the control group, the content of inorganic selenium increased form 0.04 mg/kg to 34.85 mg/kg, organic selenium increased form 0.06 mg/kg to 69.70 mg/kg, and total selenium increased form 0.103 mg/kg to 104.54 mg/kg in the leaves under Se2 treatments (p < 0.05, Figure 3B). Similarly, The content of inorganic selenium increased form 0.04 mg/kg to 6.68 mg/kg, organic selenium increased form 0.07 mg/kg to 12.61 mg/kg, and total selenium increased form 0.103 mg/kg to 19.291 mg/kg in the roots under Se2 treatments (p < 0.05, Figure 3C). In lines 313-323.

 

Q6. “For instance, under T2 treatment, the expression levels of 39 genes in the bHLH family increased by a factor of 0.43 ~ 178.03, while the expression levels of 16 genes decreased by a factor of 0.19 ~ 0.81 (Figure 7B).” Is 0.43 - 178.03 a range? Please clarify

R6# Yes, this indicates the range. I have modified this paragraph

For instance, Compared to the control group, under Se2 treatment, the expression levels of 39 genes in the bHLH family increased by 0.43 ~ 178.03 times, while the expression levels of 16 genes decreased by 0.19 ~ 0.81 times (Figure 7A). Similarly, the expression levels of 41 genes in the MYB family increased by 0.27 ~ 720.60 times, whereas the expression levels of 10 genes decreased by 0.34 ~ 0.88 times (Figure 7B). In a word, Under Se2 treatment, exceeding two-thirds of the transcription factors showed high expression. In lines 456-461.

 

We tried our best to improve the manuscript and made some changes marked in red in revised paper which will not influence the content and framework of the paper. We appreciate for editors and reviewers' warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer 4 Report

Comments and Suggestions for Authors

I wish to thank the authors for this interesting manuscript. However, I have some comments, please, find them below:

 

General comments:

Introduction:

1. It is suggested that Se biofortification is beneficial for human health (L45-48). It is necessary to specify what concentrations of Se are common in plants (without any treatments), and what amount is necessary to affect human health, and what – is toxic (for both plants and humans). It is stated in the abstract (L26) 1105.34 and 187.81 times increased Se accumulation. One may think that such an amount would be toxic for both (plants and humans), so it is necessary to draw the optimal range for Se.

 

Methods

2. It is necessary to specify the source of light (LED or something else) and its intensity. Similarly, the producing company/country should be specified for all used chemicals and equipment.

 

Discussion

3. Please, in the Discussion section, state clearly (based on obtained results) how exactly Se affects plant growth and quality of plant material (seeds and leaves), providing maximal advantage for nutritional side and plant growth dynamics.

 

4. Based on the obtained results, please, prepare a diagram/figure summarising how Se interacts with plants (enter, transport, accumulation) and what genes/TFs and metabolites are involved in these processes.

 

Author Response

Dear editor,

 

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft, the detailed corrections are listed below.

 

General comments:

Introduction:

Q1. It is suggested that Se biofortification is beneficial for human health (L45-48). It is necessary to specify what concentrations of Se are common in plants (without any treatments), and what amount is necessary to affect human health, and what – is toxic (for both plants and humans). It is stated in the abstract (L26) 1105.34 and 187.81 times increased Se accumulation. One may think that such an amount would be toxic for both (plants and humans), so it is necessary to draw the optimal range for Se.

R1# I have added relevant content in the introduction section in lines 111-115 and 65-70.

According to World Health Organization (WHO) standards, the recommended dose of selelium for adults is 55 μg/d, while the maximum tolerable adult intake without side effects is set at 400 μg/d. Cereal crops serve as the primary source of dietary selelium for humans. Nonetheless, their selelium content is typically low, ranging from 0.01 to 0.55 μg/g in cereals.

In plants, superfluous Se interferes with nutrient absorption, inhibits the formation of photosynthetic pigments, causes oxide damage, and induces genotoxic effects. Additionally, elevated Se levels above this threshold impede plant growth and development, thus reducing grain yield. High selenium concentrations can cause scorch-like damage to leaves, hinder branch development, reduce root growth, and even result in plant death.

In this study, we identified the optimal concentration of Se1 in the abstract section in lines 26-27.

 

Methods

Q2. It is necessary to specify the source of light (LED or something else) and its intensity. Similarly, the producing company/country should be specified for all used chemicals and equipment.

R2# Our expresson were carried in the greenhouse, and sunshine intensity ranges from 10000 to 15000 Lux in lines 154-155. I have designated production companies/countries for all chemicals and equipment used.

 

Discussion

 

Q3 Please, in the Discussion section, state clearly (based on obtained results) how exactly Se affects plant growth and quality of plant material (seeds and leaves), providing maximal advantage for nutritional side and plant growth dynamics.

R3# We added contents in lines 576-585.

In this study, under low Se1 treatment (0.02 g/kg Na₂SeO₃), the plant height, root length, leaf length, leaf width, and both dry and fresh weights of highland barley seedlings significantly increased. However, under Se2 treatment (0.2 g/kg Na₂SeO₃), the highland barley seedlings exhibited poor development, yellowing leaves, and inhibited growth (Figure 1A, B). Under Se1 low concentration treatment, the pigment content was not affected, and the activities of POD and APX increased in highland barley seedlings, which could clear the oxidative damage caused by MDA and H₂O₂ to seedlings. On the contrary, under Se2 high concentration treatment, the pigment content significantly decreased, and the antioxidant enzyme activity significantly decreased, which could not effectively eliminate the oxidative damage of MDA and H₂O₂ to seedlings. 

We also added, our previous experiments have shown that a concentration of 0.01-0.02 g/kg Na₂SeO₃ can promote the growth and development of highlandbarley seedlings, the concentration of 0.075-0.2 g/kg Na₂SeO₃ significantly inhibited the growth of seedlings (Figure S1). In lines 131 and 162-163.

 

 

Q4 Based on the obtained results, please, prepare a diagram/figure summarising how Se interacts with plants (enter, transport, accumulation) and what genes/TFs and metabolites are involved in these processes.

R4# We added the figure 11 in lines 697.

 

 

 

We tried our best to improve the manuscript and made some changes marked in red in revised paper which will not influence the content and framework of the paper. We appreciate for editors and reviewers' warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made the necessary and requested corrections

Author Response

We have made the necessary revisions as requested. Thank you to the editors and reviewers.

Author Response File: Author Response.docx