Protective Effects of Selenium on Wheat Seedlings under Salt Stress
Round 1
Reviewer 1 Report
The manuscript by Lan et al. deals with the protective effects of Se on wheat seedlings under salt a subject that falls within the scope of this journal. The title is fine but I suggest adding antioxidant or antioxidant activity as keyword.
An important aspect that it is omitted in the manuscript is in which form Se is added to the nutrient solution and the pH of the solution. Selenium oxidation state depends on pH and the behaviors of the different Se species that may form in the solution are different. In the same way the effects of selenium on wheat seedlings depend on the form in which selenium is supplied to the plant (See Guerrero et al. 2014 in PPB, 83: 300-307). Add as well in the discussion section (lines 307-310). Talking about Se treatment it is better to give the amount of Se added in µM units.
CAT and APX methodology are quite similar so they could be unified (ex. from line 114 to 117 and from 122 to 125 or from 119 to 121 and 128 to 131). Add the model in line 120.
Explain briefly how the methanol extract is obtained in line 134 and 143.
Lowercase letters in figures 1 and 2 are difficult to see compared with capital letters so I recommend increasing their size a little bit. In table 1, the lowercase letters from APX activity column need a space before them and also before the symbol “±”. In Table 2 total phenols content column, numbers need to be rearranged in order that units are always coincident between files as it is in Table 1.
I think that results from Total phenols, flavonoids, anthocyanin and pigments are not contents but concentrations. Revise the text and figures.
The authors discuss, from line 394 on, about the promotion of growth by Se but there are no data of dry biomass so they have only seen an effect on elongation growth.
Taking all the points into consideration, the manuscript must have a major revision before its acceptance to be published.
Reply to Reviewer 1
Comments and Suggestions for Authors
1. The manuscript by Lan et al. deals with the protective effects of Se on wheat seedlings under salt a subject that falls within the scope of this journal. The title is fine but I suggest adding antioxidant or antioxidant activity as keyword.
Response: The key word “antioxidant activity” has been added as suggested.
2. An important aspect that it is omitted in the manuscript is in which form Se is added to the nutrient solution and the pH of the solution. Selenium oxidation state depends on pH and the behaviors of the different Se species that may form in the solution are different. In the same way the effects of selenium on wheat seedlings depend on the form in which selenium is supplied to the plant (See Guerrero et al. 2014 in PPB, 83: 300-307). Add as well in the discussion section (lines 307-310). Talking about Se treatment it is better to give the amount of Se added in µM units.
Response:
(1) As you suggested, the published paper “Guerrero et al. 2014 (PPB, 83: 300-307)” has been cited as ref. no. [47], and discussed in the DISCUSSION section. [LINE 316- 320]
The added sentences now read as the following.
“According to the pH and redox potential of soil, two kinds of inorganic Se forms can be found: one is selenite, and another is selenate. Each of them exhibits different availabilities and effects to plants. In our study, sodium selenite (Na2SeO3) was treated in the acidic nutrient solution, which existed primarily as HSeO3- [47]. “
47. Guerrero, B.; Llugany, M.; Palacios, O.; Valiente, M. Dual effects of different selenium species on wheat. Plant Physiol Biochem. 2014, 83:300-307.
(2) The amount (μM) of Se treatment in the entire text and figures of revised manuscript has been added or revised as suggested. [LINE 321]
3. CAT and APX methodology are quite similar so they could be unified (ex. from line 114 to 117 and from 122 to 125 or from 119 to 121 and 128 to 131). Add the model in line 120.
Response: The methodology of CAT and APX has been unified into one paragraph as suggested. The revised paragraph now reads as the following. [LINE 116- 131]
CAT and APX activities were measured according to the method of Kato and Shimizu [32] and Nakano and Asada [33]. Briefly, 0.06 g of the latest newly expanded leaf was placed in 2 mL of sodium phosphate buffer (50 mM, pH 6.8) in an ice bath for extraction and centrifuged at 4°C and 12,000 g for 20 min. For CAT activity, the supernatant (0.2 mL) was collected, followed by the addition of 2.7 mL of sodium phosphate buffer (100 mM, pH 7.0), 0.05 mL of H2O, and 0.05 mL of H2O2 (1 M), and then mixing well. The absorbance of the sample solution at 240 nm (A240) was determined every 15 s for 1 min. A blank containing the same mixture with no enzyme extract was also measured. For APX activity, the supernatant (0.1 mL) was collected, followed by the sequential addition of 2.7 mL of potassium phosphate buffer (150 mM, pH 7.0), 0.4 mL of ethylenediaminetetraacetic acid (EDTA, 0.75 mM), 0.5 mL of H2O2 (6 mM), 0.5 mL of H2O, and 0.5 mL of ascorbate (1.5 mM) and then mixed well. The absorbance at 290 nm of the sample solution was determined every 15 s for 1 min using a spectrophotometer (Hitachi U3010, Tokyo, Japan). The blank containing the same mixture with no enzyme extract was also measured.
4. Explain briefly how the methanol extract is obtained in line 134 and 143.
Response: A brief explanation about the methanol extract has been added to LINE 134-138. The whole paragraph is as the following:
“The methanol extract for analyzing the DPPH scavenging capacity and the reducing power was prepared by adding 12 mL of 100% methanol to 0.02 g of lyophilized sample powder. The mixture was oscillated in an ultrasonic oscillator for 1 h and extracted overnight at 4°C. The mixture was then centrifuged at 3,000 rpm for 20 min, and the supernatant was collected for the following measurement.”
5. Lowercase letters in figures 1 and 2 are difficult to see compared with capital letters so I recommend increasing their size a little bit.
Response: The size of those lowercase letters in figures 1 and 2 has been increased as suggested.
6. In table 1, the lowercase letters from APX activity column need a space before them and also before the symbol “±”.
Response: A space has been added as suggested.
7. In Table 2 total phenols content column, numbers need to be rearranged in order that units are always coincident between files as it is in Table 1.
Response: The changes have been made as suggested.
8. I think that results from Total phenols, flavonoids, anthocyanin and pigments are not contents but concentrations. Revise the text and figures.
Response: The word “contents” of total phenols, flavonoids, anthocyanin, and pigments in the text and figures has been changed to “concentrations” as suggested.
9. The authors discuss, from line 394 on, about the promotion of growth by Se but there are no data of dry biomass so they have only seen an effect on elongation growth.
Response: Indeed, we did not record the dry biomass of untreated seedlings and Se-treated seedlings under different NaCl concentrations (Figure 1), therefore, the words “plant growth” has been changed to “plant morphogenesis” in LINE 417. The revised sentence now reads as the following.
“Moreover, Se supported the integrity of the membrane system in chloroplasts and/or mitochondria which might have also contributed to plant morphogenesis [55].”
10. Taking all the points into consideration, the manuscript must have a major revision before its acceptance to be published.
Response: Thank you very much for your critical evaluations of the paper and hope the queries answered will meet your approval for acceptance.
Author Response File: Author Response.JPG
Reviewer 2 Report
I read with interest the ms entitled “Protective effects of selenium on wheat seedlings under salt stress”. The authors performed several analyses to demonstrate the efficacy of Se in improving plant tolerance to salt stress, and obtained positive results in these respects. The experiment is well designed, although some important details are missing (as the form of Se used). The introduction could be improved. English in the ms needs improvement too.
Comments:
1) Please, move Triticum aestivum L. after wheat, line 15 in the abstract.
2) Line 42: Please, cite Pilon-Smits et al. 2009 (Curr opin plant Biol).
3) Line 49: if you abbreviate CAT, you must do it for peroxidase as well.
4) Line 64: Maybe better "leaf senescence"?
5) Line 93: Which form of selenium was used?
6) Figures: The letters above bars look sometimes weird, as they are very big but anyway different (e.g. in figures 2b, 2d, 3b). Please, check for this.
Author Response
Reply to Reviewer 2
I read with interest the ms entitled “Protective effects of selenium on wheat seedlings under salt stress”. The authors performed several analyses to demonstrate the efficacy of Se in improving plant tolerance to salt stress, and obtained positive results in these respects. The experiment is well designed, although some important details are missing (as the form of Se used). The introduction could be improved. English in the ms needs improvement too.
Response: We would like to thank you for your positive comments. We have asked a native English speaker to read and edit the language.
Comments:
1. Please, move Triticum aestivum L. after wheat, line 15 in the abstract.
Response: “Triticum aestivum L.” in ABSRTACT has been moved to the word after “wheat” as suggested. The revised sentence now reads as the following. [LINE 17]
“In this study, the mitigative effects of 22 μM selenium (Se) on seedlings of the wheat (Triticum aestivum L.) cultivar Taichung SEL-2 were investigated under different salt stress levels (0, 100, 200, 300, and 400 mM NaCl).”
2. Line 42: Please, cite Pilon-Smits et al. 2009 (Curr opin plant Biol).
Response: The published paper “Pilon-Smits et al. 2009 (Curr opin plant Biol)” has been cited as no. [4] in LINE 43 as suggested.
4. Pilon-Smits, E. A.; Quinn, C. F.; Tapken, W.; Malagoli, M.; Schiavon, M. Physiological functions of beneficial elements. Curr opin plant biol. 2009, 12 (3):267-74.
3. Line 49: if you abbreviate CAT, you must do it for peroxidase as well.
Response: an abbreviation of peroxidase (POD) has been added in LINE 50, and the revised sentence now reads as the following.
“According to previous research, possible protective mechanisms of Se in plants against stresses include its enhancement of antioxidant enzyme activities (peroxidase (POD), catalase (CAT), etc.) and increasing antioxidant compounds (anthocyanins, flavonoids, phenolic compounds, etc.), and these antioxidant systems thus reduce stress-induced oxidative situations [11].”
4. Line 64: Maybe better "leaf senescence"?
Response: “the death of leaves” has been changed to “leaf senescence”. The new sentence now reads as “Gradually, the toxicity of NaCl inhibits ion transport and results in leaf senescence and reduced photosynthesis [26].” (LINE 64-65)
5. Line 93: Which form of selenium was used?
Response: “Sodium selenite” was used and added to the nutrient solution. (LINE 94-96)
The new sentence now reads as “Se was added in the form of Sodium selenite (Na2SeO3, Sigma) to the nutrient solution (pH=4.6) with the treated concentration of 22 μM once the germinated seeds were cultivated in 150-mL beakers.”
6. Figures: The letters above bars look sometimes weird, as they are very big but anyway different (e.g. in figures 2b, 2d, 3b). Please, check for this.
Response: The letters above bars of Figures 1, 2, and 3 have been adjusted to a suitable size.
Author Response File: 
Author Response.docx
Round 2
Reviewer 1 Report
The manuscript has been improved but the statistic letters of the figures are too small, unfriendly.
Author Response
Reply to Reviewer 1Comments and Suggestions for Authors
The manuscript has been improved but the statistic letters of the figures are too small, unfriendly.
Response: The size of both capital and lowercase letters in figures 1-4 has been increased as suggested.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Reply to Academic Editor
By addressing the valuable feedback from reviewers the manuscript has been greatly improved. Please address typing errors:
1. Page 5 line 196 - spelling of 'concentration'.
Response: The type error 'concentration' has been changed.
2. In each of the figures and tables, rather than use of the word 'none' please indicate that the comparison is between '22 μM Se' and 'No added Se' or 'control'.
Response: Those words “none” and “Se” in all the figures and tables have been replaced by 'No added Se' and “'22 μM Se' as suggested.
Author Response File: Author Response.docx
