Selenium Enrichment of Green and Red Lettuce and the Induction of Radical Scavenging Potential

Round 1

Reviewer 1 Report

Brief summary:

The manuscript presents a study on agronomic fortification of two varieties of lettuce with different Se doses. The results show that the foliar application of Se can provide the daily Se requirements without causing cytotoxic effects.

Broad comments

Results and discussion. The authors continue to present the results in a one-way analysis, without differentiating between the main factors. If a two-way ANOVA has been performed, the results should be presented and discussed according to that design, establishing (in table format) and separating the differences between the main factors and their possible interaction (treatment x cultivar). The letters in Tables 1 and 2 and Figures 2 and 3 do not indicate differences between the two factors studied (If this differentiation appears in Fig. 4, contrarily to the statistical analysis described in 2.6)

The legend in Table 2 still does not describe its contents (The variable compared is not specified, nor are its units; the data is not compared using the column as a factor, but as independent values of it). In addition, the data presented in Table 2 are the same as those shown in Fig. 4, so this is superfluous and unnecessary.

In view of the data presented in Fig. 4, the greatest variability (Yield, DM, Leaves number and IC50) occurs between cultivars V1 and V2, so the information regarding the application of Se should be analyzed independently for each cultivar.

The conclusion regarding the contribution of Se by the cv of fortified lettuce should include a reference to the contents of Se per 100g (and the estimated data of Se provided seem to be surprisingly higher)

Specific comments.

Author Response

Reviewer 1.

Brief summary:

The manuscript presents a study on agronomic fortification of two varieties of lettuce with different Se doses. The results show that the foliar application of Se can provide the daily Se requirements without causing cytotoxic effects.

Broad comments

Results and discussion. The authors continue to present the results in a one-way analysis, without differentiating between the main factors. If a two-way ANOVA has been performed, the results should be presented and discussed according to that design, establishing (in table format) and separating the differences between the main factors and their possible interaction (treatment x cultivar). The letters in Tables 1 and 2 and Figures 2 and 3 do not indicate differences between the two factors studied (If this differentiation appears in Fig. 4, contrarily to the statistical analysis described in 2.6)

Response:

Thank you for the reviewer’s positive comment. Sorry for this mistake, statistical analysis has been checked again using a two-way ANOVA and differentiating between the main factors has been discussed.  All corrections were highlighted in yellow.

“Data reported in Fig. 2 (A, B and C) indicates that different Se levels (Se1: 0.019, Se2: 0.119, and Se3: 0.598 mg Se Plant-1) didn´t affect the yield, the DM and the number of leaves in both V1 and V2 significantly. However, the yield and the DM contents and the number of leaves of green lettuce (V1) grown under different Se levels were significantly greater in comparison to red lettuce (V2) plants grown under the same Se concentrations (Fig. 2, A, B and C). The lowest recorded yield, DM and number of leaves were observed in the red lettuce (V2) grown under Se3 level in comparison to green lettuce (V1) grown under Se3 treatment (Fig. 2, A, B and C). This is consistent with our recently published findings [21], where the green multi-leaf lettuce (V1) demonstrated superior crop productivity compared to the red multi-leaf (V2) lettuce”. (Lines 176-183 and 188-189).

“concentrations in both V1 and V2 lettuce cultivars (Table 1). However, Mg level increased significantly at Se1 treatment (3.90 ± 0.1 mg/g DM) in comparison to V2 (3.33 ± 0.1 mg/g DM). Additionally, greater concentrations of P, K and Ca were observed in V1 under Se1, Se2 and Se3 foliar applications in comparison to V2 under the same Se conditions. Moreover, Mn concentrations were higher in green lettuce (V1) than red lettuce (V3) under Se0, Se1, Se2, and Se3. P concentration enhanced significantly in V1 (9.95 ± 0.3 mg/g DM) in response to Se3 treatment in comparison to the control (Se0)”. (Lines 221-228).

The legend in Table 2 still does not describe its contents (The variable compared is not specified, nor are its units; the data is not compared using the column as a factor, but as independent values of it). In addition, the data presented in Table 2 are the same as those shown in Fig. 4, so this is superfluous and unnecessary.

Response:

Thank you for the reviewer’s comment. Table 2 was deleted as suggested by the reviewer.

In view of the data presented in Fig. 4, the greatest variability (Yield, DM, Leaves number and IC₅₀) occurs between cultivars V1 and V2, so the information regarding the application of Se should be analyzed independently for each cultivar.

Response:

Thank you for the reviewer’s positive comment. We are really sorry for this mistake. The results were statistically analyzed using two-way (treatment×cultivar) analysis of variance (ANOVA).

" The multi-leaf red lettuce extracts (V2) demonstrated higher ABTS radical scavenging capacity with IC50 values of 0.109 ± 0.01, 0.108 ± 0.003 and 0.092 ± 0.01 μg mL⁻¹ with Se1, Se2 and Se3 treatments respectively, in comparison to multi-leaf green lettuce extracts (V1). However, ABTS radical scavenging potential of V2 remained unaffected in response to all Se applications. In our recent published results the red lettuce (V2) showed the best radical scavenging activity against the DPPH radical [21]. The significantly greater anti-oxidant potential of V2 in comparison with V1 might be attributed to the presence of an-thocyanins, such as cyanidin 3-O-galactoside, which was only detected in V2 lettuce plants [21]. The IC50 of ascorbic acid (positive control) was 0.0013 mg/mL." (Line 273-281).

All corrections were highlighted in yellow in the references list.

The conclusion regarding the contribution of Se by the cv of fortified lettuce should include a reference to the contents of Se per 100g (and the estimated data of Se provided seem to be surprisingly higher)

Response:

Thank you for the reviewer’s positive comment. References for the calculations of Se daily requirements were included in the text (Line 206) and the references list. All corrections were highlighted in yellow.

1. Zou, C.; Du, Y.; Rashid, A.; Ram, H.; Savasli, E.; Pieterse, P.J.; Ortiz-Monasterio, I.; Yazici, A.; Kaur, C.; Mahmood, K.; Singh, S.; Le Roux, M.R.; Kuang, W.; Onder, O.; Kalayci, M.; Cakmak, I. Simultaneous biofortification of wheat with Zinc, Iodine, Selenium, and Iron through foliar treatment of a micronutrient cocktail in six countries. J. Agric. Food Chem. 2019, 67, 8096–8106.
2. Ram, H.; Rashid, A.; Zhang, W.; Duarte, A.P.; Phattarakul, N.; Simunji, S.; Kalayci, M.; Freitas, R.; Rerkasem, B.; Bal, R.S.; Mahmood, K.; Savasli, E.; Lungu, O.; Wang, Z.H.; de Barros, V.L.N.P.; Malik, S.S.; Arisoy, R.Z.; Guo, J.X.; Sohu, V.S.; Zou, C.Q.; Cakmak, I. Biofortification of wheat, rice and common bean by applying foliar zinc fertilizer along with pesticides in seven countries. Plant Soil 2016, 403, 389–401.

Regarding the calculations of Se daily requirements and the percentage of covering this requirements. Although the recommended dietary allowance of 70 µg day^-1 for adult men and 60 µg day^-1 for adult women is a minimum. We calculated again according to the reviewer comment that consuming 75-90 and 100 g fresh weight from V1 and V2 will cover the Se requirement by 100%.

“Accordingly, both V1 and V2 lettuce cultivars grown under Se3 foliar application condition can cover the daily requirement for adult men by approximately (100%) and (85% to 100%), respectively by consuming 75-90 and 100 g fresh weight from V1 and V2, respectively”. (Lines 20-21).

“Furthermore, both V1 and V2 lettuce cultivars can participate to Se requirements with (78.4 – 94.4 µg Se day-1) and (59.6 – 71.6 µg Se day-1), respectively by consuming 100 g fresh weight lettuce heads grown under Se3 conditions [29,30]. In this regard, we rec-ommend that V1 and V2 can cover the dietary allowance for adult men with consuming 75-90 and 100 g fresh weight from V1 and V2 grown under Se3 treatment, respectively”. (Lines 204-209).

Author Response File: Author Response.docx

Reviewer 2 Report

my previous suggestions have been addressed and no further actions are required from this side.

Author Response

Reviewer 2

Comments and Suggestions for Authors

my previous suggestions have been addressed and no further actions are required from this side.

Response:

Thank you for the reviewer’s positive comment.

Round 2

Reviewer 1 Report

Brief summary:

The manuscript is clear, and presented in a well-structured manner. It addresses a study on agronomic biofortification of two cv of lettuce with Se. The results show that the foliar application of Se can provide the daily requirements of Se without causing cytotoxic effects.

Broad comments

Introduction is clear and concise, making explicit the hypothesis and the work objectives.

Material and Methods comprehensively describes the conditions of the experiment, crop management and methods of analysis.

Results are interesting and reproducible, however, the effect of the main factors is not clearly appreciated, nor the realization of the two-way ANOVA. Discussion addresses aspects related to the contributions of Se from fortified lettuces and their effects on health.

Conclusions are consistent with the evidence and arguments presented.

The references are current and collect most of the information published in the topic

Specific comments

Line 210; 216; 264. Fig 2; 3 and 4.. The values would be more easily interpretable, if the significance due to the effect of the application of Se were represented with different types of letters for each cv (for example, uppercase for V1 and lowercase for V2.)

Line 246. Table 1: The values would be more easily interpretable, if the significance due to the effect of the application of Se are represented with different types of letters for each cv (for example, uppercase for V1 and lowercase for V2.)

Author Response

Prof. Dr. Wilfried Rozhon

Prof. Dr. Sabine von Tucher

Special Issue Editors

Horticulturae MPDI

Dear Editor,

Thank you very much for editing our manuscript entitled "Selenium enrichment of green and red lettuce and the induction of radical scavenging potential" which was submitted to Horticulturae MPDI.

We have revised the manuscript carefully again according to the Reviewer’s comments as you can see in “Responses to the Reviewers’ Comments”. Please find my responses to the reviewers’ comments below.

I hope that my revised manuscript is now fully acceptable for publication in Horticulturae MPDI.

I look forward to receiving positive decision from you.

Sincerely,

Prof. Dr. Karl H. Mühling,

Institute of Plant Nutrition and Soil Science

Kiel University

Hermann-Rodewald-Str. 2, 24118 Kiel, Germany

Email: [email protected]

Responses to reviewers’ comments

Reviewer 2. Broad comments

Introduction is clear and concise, making explicit the hypothesis and the work objectives.

Material and Methods comprehensively describes the conditions of the experiment, crop management and methods of analysis.

Results are interesting and reproducible, however, the effect of the main factors is not clearly appreciated, nor the realization of the two-way ANOVA. Discussion addresses aspects related to the contributions of Se from fortified lettuces and their effects on health.

Conclusions are consistent with the evidence and arguments presented.

The references are current and collect most of the information published in the topic

Response:

Thank you for the reviewer’s positive comment.

Specific comments

Line 210; 216; 264. Fig 2; 3 and 4.. The values would be more easily interpretable, if the significance due to the effect of the application of Se were represented with different types of letters for each cv (for example, uppercase for V1 and lowercase for V2.)

Line 246. Table 1: The values would be more easily interpretable, if the significance due to the effect of the application of Se are represented with different types of letters for each cv (for example, uppercase for V1 and lowercase for V2.)

Response:

With respect to the reviewer valuable opinion, we think that if we present the Figures 2, 3 and 4 and Table 1 in the way suggested by the reviewer it would be hard to statistically judge the differences between both cultivars (V1 and V2) under the same Se treatment. Thus, we prefer to keep the Figures 2, 3 and 4 and Table 1 in the same way. In support, we used the same statistical analysis approach in our recently published paper: Abdalla, M.A.; Li, F.; Wenzel-Storjohann, A.; Sulieman, S.; Tasdemir, D.; Mühling, K.H. Comparative metabolite profile, biological activity and overall quality of three lettuce (Lactuca sativa L., Asteraceae) cultivars in response to sulfur nutrition. Pharmaceutics 2021, 13, 713.

In conclusion, we think that the way of presenting the data (including the letters in the current form) can help the reader to compare between V1 and V2 cultivars under the same Se level as well as between the same cultivar under different Se treatments.

If we present the letters as suggested by the reviewer (uppercase for V1 and lowercase for V2), we will differentiate between the same cultivars under different Se treatments, but cannot show the differences between cultivars (V1 and V2) under the same Se treatments.

Author Response File: Author Response.docx