Iodine Enhances the Nutritional Value but Not the Tolerance of Lettuce to NaCl

Round 1

Reviewer 1 Report

It is requested to improve the introduction and discussion, in addition to updating the citations (2018-2022).

There are formatting corrections to be made to the manuscript.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

R1: It is requested to improve the introduction and discussion, in addition to updating the citations (2018-2022).

A1: We improved the “Introduction” and “Discussion” sections, also updating citations.

R2: There are formatting corrections to be made to the manuscript.

A2: We thank this Reviewer for his/her suggestions and corrections. We modified the manuscript accordingly.

Reviewer 2 Report

The manuscript is very well written. It has a theoretical foundation and its hypothesis and objectives are very well defined, clear. Some issues were raised by me in the Material and Methods item, but nothing that would harm the manuscript. They are considerations to improve the information presented. The results are very good and can contribute to other researches in the field of vegetable quality/biofortification.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

R1: The manuscript is very well written. It has a theoretical foundation, and its hypothesis and objectives are very well defined, clear. Some issues were raised by me in the Material and Methods item, but nothing that would harm the manuscript. They are considerations to improve the information presented. The results are very good and can contribute to other researchs in the field of vegetable quality/biofortification.

A2: We thanks the Reviewer for his/her appreciation to manuscript. We improved the manuscript according to his/her suggestions and comments as reported in pdf file. In the improved version of manuscript, we specified that all measurements were carried out at the harvesting, which occurred at 60 days after sowing, when plants had sufficiently expanded leaves and a higher postharvest quality should be expected.

Reviewer 3 Report

The article “Iodine enhances the nutritional value but not the tolerance of lettuce to NaCl stress“ is well–written and interesting to read. However, some aspects are not entirely clear and need to be explained more in detail.

-          First in the material & methods part, the iodine and salt application is not clear. Did the pots receive the treatment once or with every nutrition? If they received it once is it correct that 50 mL nutrient solution contained 2.7µg I. If it was applied with every nutrient application it is important to know how often the plants received this treatment. The total amount of I/pot should be clear. I had the same problem with the salt that I did not know if every nutrient solution contained NaCl in the NaCl treatments.
How long did the plants grow till harvest?

-          How did you define “Stress” in your trial? There was no yield effect at all and photosynthesis was not affected by NaCl. Why did you think your plants were exposed to mild stress? This should be explained because otherwise it is not clear why the plant should react in any way.

-          A treatment receiving only KIO₃ is unfortunately missing. With the given design of the trial it is not possible to separate the effect of I.

-          A clear aim of the study is missing. If it is the intention to look if plant quality can be increased due to I application than quality parameter should be defined and addressed. In this context it would be interesting to know if the plants took up I and if it was increased in the plant material. For the parameter that were determined there is no explanation if the observed increase is of relevance. If the focus was on the point if lettuce can be grown on salt-affected soils when I is applied, than the study does not really give an answer because it would have been necessary to work with a NaCl concentration where lettuce is negatively affected. Otherwise, I think, it is not really possible to answer this question.

-          The conclusion that the nutraceutical value can be increased by Iodine need to be explained more in detail.

-          The English needs a revision there a several little mistakes such as:

Line 17 must be of instead of pf

Line 19 … subjected to moderate salt stress provoked by NaCl alone or by NaCl in combination with iodine.

Line 43 … it is not clear…

Line 52 blank is missing before improves

Line 63 what is a mixed composted soil?

Line 84 + 88 one bracket too much  

Line 93 30´? What does that mean?

Line 94 missing blank after 2.3

Line 135 missing blank between at and 595

Table 3 include “antioxidant capacity” before FRAP

Line 202 ff this paragraph is hard to understand. Moreover, it is not clear why elevated CO₂ was studied.

Figure 2 Letters are missing partly (a-f)

Line 239 blank is missing behind CO₂

Line 265 two-times other

Line 294 of the contribute?

Line 300 PCR is not explained

Author Response

Reviewer 3

The article “Iodine enhances the nutritional value but not the tolerance of lettuce to NaCl stress“ is well–written and interesting to read. However, some aspects are not entirely clear and need to be explained more in detail.

R1: First in the material & methods part, the iodine and salt application is not clear. Did the pots receive the treatment once or with every nutrition? If they received it once is it correct that 50 mL nutrient solution contained 2.7µg I. If it was applied with every nutrient application it is important to know how often the plants received this treatment. The total amount of I/pot should be clear. I had the same problem with the salt that I did not know if every nutrient solution contained NaCl in the NaCl treatments.

A1: Plants were fertilized weekly until the harvest providing 50 mL of nutrient solution in which NaCl or NaCl + KIO₃ were dissolved, while the control only received nutrient solution. This point has been clarified in Material and methods.

R2: How long did the plants grow till harvest?

A2: Plants have been harvested at 60 days after sowing. This information has been added in Material and methods.

R3: How did you define “Stress” in your trial? There was no yield effect at all and photosynthesis was not affected by NaCl. Why did you think your plants were exposed to mild stress? This should be explained because otherwise it is not clear why the plant should react in any way.

A3: We thank the Reviewer for his/her useful comments. We agree with all the observations. We removed “stress” from the title and modified all manuscript accordingly.

R4: A treatment receiving only KIO3 is unfortunately missing. With the given design of the trial it is not possible to separate the effect of I.

A4: We thank the Reviewer for his/her appropriate comment. Unfortunately, we do not have a treatment with iodine alone that should allow separating the effect of iodine. Our purpose was to evaluate if iodine can improve the salt tolerance in lettuce through an enhancement in photorespiration and bioactive compounds, including antioxidants. We agree that our experimental trial would be better by including the treatment with the only KIO3; but at the same time, we believe that the evidence provided in this study may help meet the purpose of the research (even if it is not possible to separate the effect of I, as rightly pointed out by Reviewer).   

R5: A clear aim of the study is missing. If it is the intention to look if plant quality can be increased due to I application than quality parameter should be defined and addressed. In this context it would be interesting to know if the plants took up I and if it was increased in the plant material. For the parameter that were determined there is no explanation if the observed increase is of relevance. If the focus was on the point if lettuce can be grown on salt-affected soils when I is applied, than the study does not really give an answer because it would have been necessary to work with a NaCl concentration where lettuce is negatively affected. Otherwise, I think, it is not really possible to answer this question.

A5: We thank the Reviewer for his/her appreciable comment. Unfortunately, we are not the equipment for I determination in vegetables. However, we modified the manuscript according to Reviewer’s criticisms, also specifying that bioactive compounds (chl, car, polyphenols, and soluble proteins) were used as indicator of vegetable quality.

R6: The conclusion that the nutraceutical value can be increased by Iodine need to be explained more in detail.

A6: We have improved this point.

R7: The English needs a revision there a several little mistakes such as:

Line 17 must be of instead of pf

A7: Done

R8: Line 19 … subjected to moderate salt stress provoked by NaCl alone or by NaCl in combination with iodine.

A8: Done

R9: Line 43 … it is not clear…

A9: This issue has been deleted in the new version of manuscript.

R10: Line 52 blank is missing before improves

A10: We have corrected

R11: Line 63 what is a mixed composted soil?

A11: It means a peat soil. This important issue has been clarified in Material and Methods.  

R12: Line 84 + 88 one bracket too much 

A12: Corrected

R13: Line 93 30´? What does that mean?

A13: 30 minutes

R14: Line 94 missing blank after 2.3

A14: Done

R15: Line 135 missing blank between at and 595

A15: Corrected

R16: Table 3 include “antioxidant capacity” before FRAP

A16: We have replaced “FRAP” with “Antioxidant capacity”

R17: Line 202 ff this paragraph is hard to understand. Moreover, it is not clear why elevated CO2 was studied.

A17: In Material and Methods (paragraph 2.2) we explain why we performed measurements to elevated CO₂

R18: Figure 2 Letters are missing partly (a-f)

A18: We have corrected

R19: Line 239 blank is missing behind CO2

A19: Corrected

R20: Line 265 two-times other

A20: Corrected

R21: Line 294 of the contribute?

A21: We have modified this sentence

R22: Line 300 PCR is not explained

A22: We have modified this sentence

Reviewer 4 Report

Dears Autors,

The reviewed article concerns an important issue which is the biofortification of plants in iodine. Biofortification of the main crops with micronutrients using agrotechnical solutions is a cost-effective and sustainable approach to solving deficiencies of these elements in the human diet. The possibility of transporting iodine to the edible parts of plants has been confirmed for various crops (leaf plants such as lettuce, cereals, nectarines and plums, tomatoes, strawberries). The most important studies concerned the effectiveness of enriching plants with various levels of iodine, as well as the methods of iodine application best suited to obtain the biofortification effect. This work pays particular attention to the efficiency of iodine biofortification under stressful conditions such as salinity.

The first question that arises for the authors is "Why was not an iodine-only fertilization experiment planned?"

In the opinion of the reviewer, some important information is missing in chapter 2.1, which should be supplemented.

The authors write that the seedlings were transplanted into pots. Please complete the information about sowing seeds and the time of replanting. Moreover, the exact composition of the nutrients solution was not given, which seems to be a necessity. Please also provide information on the number of fertilization treatments and the date of harvest (in what week after planting the seedling in the pot, the harvest took place).

In the Results chapter, there is no information about the appearance of the harvested lettuce heads - did the applied dose of salt and salt + iodine affect their appearance? Were any discolorations visible, or was the average weight of the lettuce heads different between the experiments? The authors should consider the total lettuce yield in each experiment.

In chapter 3.1, when discussing soil parameters, there is no comment on the availability and mobility of iodine in such soil. In addition, the content of soil organic matter should be given because it has an impact on the uptake efficiency of this element by plants.

Also note on statistics. In tables, the results that do not differ statistically do not have letters, while in the opinion of the reviewer, the letter "a" should be entered everywhere. This is due to the fact that the authors write under the table "Different letters indicate significant differences among treatments". Additionally the letter "a" is assigned to the lowest scores while this convention is broken for the total polyphenol content.

Additionally, below are some suggestions that I hope will help improve this work.

1. line 17 - is "pf" should be "of"

2. line 41 - is "I +" should be "I-"

3. line 68 - space is missing (it is "53 mg I", it should be "53 mg I")

4. line 80 - space is missing (it's "m-2s-1" should be "m-2s-1")

5. line 113 - missing parenthesis

6. line 116 - missing spaces at the end of the line

7.line 120 - space is missing (is "31.6 M-1cm-1" should be "31.6 M-1 cm-1")

8.line 135 - space is missing ("at595" is "at 595")

9. line 136 - missing spaces at the end of the line

10. line 162 - proposes to add "FRAP" next to "antioxidant capacity"

11. lines 160-168 - no comment on the content of flavonoids

12. Figure 2 - no letters signing subsequent charts

13. Figure 2 - graph e) filled bars (measurement to ambient CO2) is there really a statistical difference between the experiments? If so, it seems that the values for the experiment with NaCl and NaCl + KIO3 fertilization are lower than for the control and not higher as suggested by the letters above the bars

14. Figure 2 – graph f) Please explain why small differences between the values for ambient CO2 are statistically significant, while much larger differences of the same parameter measurements to elevated CO2 do not show a statistical difference

15. Lines 244-247 - please provide relevant references

16. Lines 253-255 - Reference 31 cited relates to fertilization with selenium and not with iodine

Best regards,

Reviewer

Author Response

Reviewer 4

The reviewed article concerns an important issue which is the biofortification of plants in iodine. Biofortification of the main crops with micronutrients using agrotechnical solutions is a cost-effective and sustainable approach to solving deficiencies of these elements in the human diet. The possibility of transporting iodine to the edible parts of plants has been confirmed for various crops (leaf plants such as lettuce, cereals, nectarines and plums, tomatoes, strawberries). The most important studies concerned the effectiveness of enriching plants with various levels of iodine, as well as the methods of iodine application best suited to obtain the biofortification effect. This work pays particular attention to the efficiency of iodine biofortification under stressful conditions such as salinity.

R1: The first question that arises for the authors is "Why was not an iodine-only fertilization experiment planned?"

A1: We thank the Reviewer for his/her appropriate comment. Unfortunately, we do not have a treatment with iodine alone that should allow separating the effect of iodine. Our purpose was to evaluate if iodine can improve the salt tolerance in lettuce through an enhancement in photorespiration and bioactive compounds, including antioxidants. We agree that our experimental trial would be better by including the treatment with the only KIO3; but at the same time, we believe that the evidence provided in this study may help meet the purpose of the research.   

R2: In the opinion of the reviewer, some important information is missing in chapter 2.1, which should be supplemented.

The authors write that the seedlings were transplanted into pots. Please complete the information about sowing seeds and the time of replanting. Moreover, the exact composition of the nutrients solution was not given, which seems to be a necessity. Please also provide information on the number of fertilization treatments and the date of harvest (in what week after planting the seedling in the pot, the harvest took place).

A2: Seedlings were purchased from a nursery and the transplant took place about at 20 days after sowing. We added this information in the text. We also reported the exact composition of the nutrient solution in the new version of manuscript and added information about the number of fertilization treatments and the date of harvest.

R3: In the Results chapter, there is no information about the appearance of the harvested lettuce heads - did the applied dose of salt and salt + iodine affect their appearance? Were any discolorations visible, or was the average weight of the lettuce heads different between the experiments? The authors should consider the total lettuce yield in each experiment.

A3: No difference due to salt treatment has been observed among treatment. This information has been added in the section Results (3.1 paragraph). We did not consider the root weigh because it has been difficult to remove the soil around the root; moreover, many rootlets were dispersed in the soil, underestimating the root biomass.

R4: In chapter 3.1, when discussing soil parameters, there is no comment on the availability and mobility of iodine in such soil. In addition, the content of soil organic matter should be given because it has an impact on the uptake efficiency of this element by plants.

A4: No information on the availability and mobility of iodine in soil is reported because we are not equipped for these determinations, however, the organic carbon and C/N ratio in soil have been added in the new version of manuscript.

R5: Also note on statistics. In tables, the results that do not differ statistically do not have letters, while in the opinion of the reviewer, the letter "a" should be entered everywhere. This is due to the fact that the authors write under the table "Different letters indicate significant differences among treatments". Additionally the letter "a" is assigned to the lowest scores while this convention is broken for the total polyphenol content.

A5: We have corrected according to Reviewers’ comments and suggestions.

R5: Additionally, below are some suggestions that I hope will help improve this work.

1. line 17 - is "pf" should be "of"

Corrected

2. line 41 - is "I +" should be "I-"

We deleted this sentence

3. line 68 - space is missing (it is "53 mg I", it should be "53 mg I")

Added

4. line 80 - space is missing (it's "m-2s-1" should be "m-2s-1")

Added

5. line 113 - missing parenthesis

Added

6. line 116 - missing spaces at the end of the line

Added

7.line 120 - space is missing (is "31.6 M-1cm-1" should be "31.6 M-1 cm-1")

Added

8.line 135 - space is missing ("at595" is "at 595")

Added

9. line 136 - missing spaces at the end of the line

Added

10. line 162 - proposes to add "FRAP" next to "antioxidant capacity"

Added

11. lines 160-168 - no comment on the content of flavonoids

Added

12. Figure 2 - no letters signing subsequent charts

Added

13. Figure 2 - graph e) filled bars (measurement to ambient CO2) is there really a statistical difference between the experiments? If so, it seems that the values for the experiment with NaCl and NaCl + KIO3 fertilization are lower than for the control and not higher as suggested by the letters above the bars

We have rearranged letters indicating significant differences reporting the “a” letter for lower values.

14. Figure 2 – graph f) Please explain why small differences between the values for ambient CO2 are statistically significant, while much larger differences of the same parameter measurements to elevated CO2 do not show a statistical difference

At elevated CO₂, differences are not significant due to a greater error bars than those reported at ambient CO₂.

15. Lines 244-247 - please provide relevant references

Done

16. Lines 253-255 - Reference 31 cited relates to fertilization with selenium and not with iodine

We have deleted this reference

Round 2

Reviewer 3 Report

Dear authors,

your manuscript has been improved a lot but I think a thorough English editing would improve your paper a lot. As I´m also no native English speaker I´m not always sure how to express best.

Here are some morre English mistakes I recognized, but there are more in your manuscript

L 17 "an improvement"  or ".. by increasing photorespiration and the content of antioxidants"

L 26 compound instead of compounds

L 41 while severe salinity...

L 58 "it is not clear.." or "it is not well understood..."

L 67 "in particular the application of..."

L 70 effect of salinity on moderate ...

L 71 "sultivated for and consumed in human diet"

L 73 a higher

L 88 following growth conditions

L 185 harvesting of control plants

L 200 caused a reduction by 74% in ..

L 202 by 95% and 21%...

L 267 activated by salt stress

L 293 what do you mean by sensitive in this context? in sensitive plants?

L 294 induced

I think a good English revision would increase the readability of the manuscript a lot.

Author Response

A: We thanks the Reviewer for his/her suggestion and we improved manuscript by having English reviewed by an expert.

Reviewer 4 Report

Dear Authors,
thank you for comprehensive answers to the questions asked and for clarifying all doubts that arose while reading the first version of the manuscript.

Kind regards,

Reviewer

Author Response

A: We thanks the Reviewer for his/her appreciation of the revised version.