Red to Blue Light Ratio and Iron Nutrition Influence Growth, Metabolic Response, and Mineral Nutrients of Spinach Grown Indoors

Round 1

Reviewer 1 Report

General comments: The manuscript entitled "The Effect of Different Red and Blue Light Ratios on Growth, Metabolic Response, and Mineral Nutrients of Spinach Biofortified with Iron" investigated the effects of three ratios of blue and red light on the growth of two cultivars of spinach. The growth parameters, chlorophyll content, mineral elements, soluble sugar, antioxidant capacity, and total phenolic content were measured. The manuscript is well written. The results are described precisely. This study provides us with some new insights into the production of leafy vegetables indoors. I recommend that the manuscript can be published in the journal after some minor modifications. My major concerns are as follows:

1. Line20-21 Leaf length, width, and number and plant biomass

Revised as---The length, width, and number of leaf and plant biomass

2. Why the authors choose Fe Chelate as Fe source? This should be explained in the text.

 

3. Why the experiments were set with two replicates? We know that scientific studies generally contain three or more replicates.

 

4. The discussion section is too long, I suggest to decrease the repetitive description of the results.

 

 

5. Reference: the format should follow the requirements of the journal. Pleas recheck them carefully. Scientific name of biological species should be italic font. E,g, Reference 1 : Spinacia Oleracea should be italic Spinacia Oleracea 

Author Response

Dear Reviewer,

Thank you for taking the time to read our article and for your meaningful comments. Please see the attachment. 

Sincerely, Authors

Author Response File: Author Response.pdf

Reviewer 2 Report

This extensive study entitled “The Effect of Different Red and Blue Light Ratios on Growth, Metabolic Response, and Mineral Nutrients of Spinach Biofortified with Iron” by Vaštakaitė-Kairienė et. al. studied the effect of  different light on growth, metabolic response, and accumulation of mineral nutrients in Spinach. Leafy vegetables contain substantial health-promoting nutrients although they are not enough to fulfill the recommended dietary allowance (RDA for human health. Any strategy to increase the nutrient level in food crops are appreciated which can provide RDA for better human health. Therefore, this study has great value. In my view, this study is logically planned and executed, and the results are appropriately interpreted. However, I have only one comment that needs to be addressed prior to the final publication.

 

 

Abstract

The abstract is too big, please make it short

Line 24-26: Please revise the sentence.

Introduction:

Citations are not in appropriate order, please revise the whole introduction section.

Line 31-36: Please provide the content of specific vitamins and minerals, phytochemicals, and bioactive compounds in Spinach with appropriate reference.

Line 101: Did you study light quality/quantity or both, please mention in the objectives.

Line 103: What is the rationale behind the analysis of saccharides, total phenolic content, and antioxidant capacity?

Materials and Methods

Line 108: Are they cultivar or variety? Why did you choose two spinach cultivars?

 

Line 108: Did you use any certified reference materials (CRM) during your analysis? If yes, please mention the certified value and your observed value.

Minor Comments:

 

Some data can be presented as bar/line graph.

Author Response

Dear Reviewer,

Thank you for taking the time to read our article and for your meaningful comments. Please see the attachment. 

Sincerely, Authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Review report by Prof. Nigussie Dechassa

Title (Lines 1–3)

 

The Effect of Different Red and Blue Light Ratios on Growth, Metabolic Response, and Mineral Nutrients of Spinach Biofortified with Iron

 

In this title, the definite article “The” is a wasteword. This is because it does not add any value except lengthening it. Therefore, delete it.

 

In this Title, what do you mean by the noun phrase “Mineral nutrients”?

Are you referring to contents or concentrations of the mineral nutrients in the spinach leaf dry matter?

What does “Biofortified with Iron” mean?

Biofortification is increasing the concentration of microniutrtrents in plants (seeds or leaves) by applying nutrients for enhanced uptake by plants during growth.

Therefore, it is the nutrient taken up by the plant during growth that is considered biofortified rather than the nutrient that is applied in the medium of growth (nutrient solution).

Then, how can applying Fe in a nutrient solution be termed as biofertification?

Therefore, this Title is not descriptive enough of the research done!

 

Suggested Title: Effect of Different Red and Blue Light Ratios on Growth, Metabolic Response, and Concentration of Mineral Nutrients in Spinach Plants Supplied with Iron in a Hydroponic Solution

 

Abstract (Lines 11–28)

Ana abstract should stand alone by including brief rationale, objective, methodology, key results, conclusion, and implication of the results. However, some of these key elements are missing in this abstract.

What was the objective of the study?

This should have come after the problem statement (lack of information (line 15?). To fortify spinach plants with iron by manipulating Fe concentration in a nutrient solution and rations of Red and Blue lights from light-emitting diodes (LEDs)??

 

In indicating the methods in brief, what was the spinach cultivar used for the experiment? How many? One or two or more? What was the design of the experiment? What method of analyzing the results was used?

 

Lines 16–17:

………………… when nutrient solutions are biofortified with particular mineral nutrients

Can the term biofertilization be applied here? Is it not better to write “…………when the nutrient solution or when the spinach plant was applied with mineral nutrients?”

Indicate the type hydroponic solution used. Was it the standard Hoagland solution or any other solution?

 

Lines 16–17:

…..Leaf length, width, and number and plant biomass generally decreased as the R:B light ratio decreased, leading to a high concentration of Fe in hydroponic solution.

What can be the relationship between the decrease or increase in R:B light ratio and the concentration of Fe in the hydroponic solution?  I would have thought the increased plant biomass and leaf length and width would rather result from increased uptake of Fe from the nutrient solution. Thus, the concentration of Fe in the nutrient solution would rather decrease. This is not clear!

Lines 22–23:

“A higher Fe concentration in the solution increased contents of some other mineral nutrients but this depended on the R:B and cultivar”. But you did not indicate the cultivars you used in the abstrcat. Were there different cultivars or only one cultivar in the treatment?

 

What were these nutrients? Where did these nutrients come from?

By how much percentage or how many fold was the increase?

 

 

Lines 23–25:

For example, the contents of Zn generally increased with increasing Fe concentration in the solution but decreased the Cu content (or but the Cu content decreased?), especially in ‘Space F1’.

What is “Space F1”? It was not described here? Is it a cultivar?

By how much was the contents of Zn generally increased with increasing Fe concentration?

What does “generally” means here?

The key results in the abstract should be quantified.

Generic description of results in the abstract would make the abstract understandable.

Conclude the abstract by describing the key results and giving the implications.

Lines 26–28 (Implication?)

“These results suggest that the overall quality of spinach might improve with lighting and Fe bio fortification strategies and thus increase the sustainability of indoor crop production.”

But, the authors did not place here any quantified data (mean values) as affected by the treatments. Thus, which key results warrant this implication? How can we accept the implication without knowing the key results obtained from this study?

In general this abstract misses the following important contents:

Clear objective

·       Briefly stated clear methodology (including clear treatments, for example, no cultivars mentioned)

·       Experimental design

·       Clear statistical methods used to analyse the data

·       Clear results (findings) no quantification of the key resultys obtained

·       Clear conclusion

 

 Keywords (Lines 29 & 30)

The words “Iron” and “mineral nutrients” are already listed in the Title. Therefore, these two words should not be listed again as keywords.

In addition, there is no much information about “biofertification” of iron in spinach in the results and discussion section of this study. Therefore, I do not think this word should be listed as a keyword.

In the list of keywords, include, the botanical name  Spinacia oleracea

Include also the keywords relative chlorophyll content (SPAD), soluble sugars, plant biomass, and leaf growth.

1.   Introduction

Lines 112 & 113

The Introduction is generally written well.

But, describe the problem statement clearly.

This problem statement must be related to of micronutrient (Fe) deficiency in spinach leaves grown under such conditions (artificial light or indoor farming?). Is the problem of Fe deficiency in spinach plants obtained from indoor farming serious enough to warrant the research?

Elaborate on this well.

Other sections of the Introduction are well written.

Materials and Methods (Line 123)

First describe the nutrient solution (was it the standard Hoagland or modified one?)

Describe the two cultivars of spinach used?

Next, before describing the growth conditions (procedure) as done under the sub-heading 2.1 (Line 105), it is important to describe the treatments. What were the Fe treatments?

How many Fe concentrations and which concentrations in the nutrient solution?

Did the treatment include control or nil concentration?

What were the light treatments? How many and which ratios?

What were the cultivar treatments? How many and which cultivars were used?

It appears that there were three factors at different levels, i.e., Fe concentration in the nutrient solution, spinach cultivars, and light photons (Blue-Red ratios)

What was the design of the treatments?

How were the treatments arranged?

Were the treatment arranged factorially?  In fact, the two-way analysis of described in lines 224 to 228 imply this.

Line 110

…”were own”.  Change it to …”were sown”.

2.     Results (Line 229)

For understanding the levels of significance and non-significance of the two-way analysis of variance indicated above under the sub-heading 2.7. Statistical analyses, it is important that the authors show one table of the mean squares for all variables studied showing the degrees of freedom and the level of significance or non-significance.

This means it is not sufficient to show only the mean values of the variables.

 

Line 231 & 232

“There was no effect of the R:B light ratio on the SPAD index of either spinach cultivar 231 studied (Table 2)". What does either spinach cultivar mea here? Do you mean both cultivars, which data show in fact?

In fact, Table 2 shows only the mean values of SPAD of both cultivars, which were not affected by R:B light ratio. At what levels of significance were the values unaffected?

If a reader wants to know this, it is important to show the Table of mean square as indicated above.

By the way, what is the unit of SPAD index?

 

Line 234:

“The leaf length and width generally decreased with R:B light ratio”.

 Did the decrease in leaf length occur with increase or decrease in R:B light ratio?

Write clearly.

What does “generally” mean here?  The authors need to avoid such generic terms in scientific writing and quantify the increase or decrease. For example, indicate by how much percentage or how many fold leaf length decreased compared to the control or any other treatment.

 

Why did the authors present a table to for each cultivar separately? Was there no interaction of cultivar and the light or/and Fe treatment on the variables presented in each column? Unless one sees a table of mean squares, how is it possible to know whether there was interaction effect?

Lines 264 & 265

“Spinach uptake of macronutrients P, Ca, and K (but not Mg) was also influenced by the Fe concentration in the hydroponic solution”.

Here, the authors express concentrations (content) of mineral nutrients in the dry matter of spinach leaves as synonymous with uptake.  It is important to know that these two terms connote different concepts, Uptake refers to total uptake of a mineral nutrient by a plant biomass at harvest. Therefore, the authors need to maintain consistency in the use of terms.

 

3.     Discussion (line 37)

 

Growth (Line 38)

It appears that the authors have not focused their discussion on their own findings. Rather the discussions appears to be a sort of literature review.

“For example, one of the key findings was that B:R light ration did not influence leaf length and leaf width, but the concentration of Fe in the nutrient solution affected these variables”. Why may have these results occurred? Specula and give possible causes by supporting with evidence from previous research.

 

What is more, for example, Lines 231 to 234, “there was no effect of the R:B light ratio on the SPAD index of one spinach cultivar). However, SPAD index was greater than the control treatment when spinach ‘Space F1’ was grown under an R:B ≤ 3:1 and ≥ 5 mg L–1 233 of Fe”. Where is the discussion on these results?

Lines 252–274

“The Fe content in hydroponic solution influenced the uptake of micronutrients Zn and Cu in both spinach cultivars. Generally, the contents of Zn increased with increasing Fe concentration in the solution. In both cultivars, the highest contents of Zn were in plants grown in solution with 15 mg L–1 of Fe, regardless of the R:B light ratio. Compared to the control, the Zn content increased by 15% in ‘Corvair F1’ and 49% in ‘Space F1’ when spin-ach was grown with the highest Fe concentration. In contrast, the 15 mg L–1 of Fe in the solution somewhat decreased the Cu content, especially in ‘Space F1’. Spinach uptake of macronutrients P, Ca, and K (but not Mg) was also influenced by the Fe concentration in the hydroponic solution. The content of P in ‘Corvair F1’ plants increased, regardless of the R:B ratio, with an elevated Fe concentration in the solution. Similarly, Ca content in  ‘Corvair F1’ plants grown in a solution with 15 mg L–1 of Fe was up to 77% greater than plants in the control treatment”.  Where is the discussion on these results? Or the discussion is not robust.

 

Lines 379–383

“Our results demonstrated that while the R:B light ratio had no consistent effect on the SPAD index (the relative chlorophyll content), higher doses of Fe  in the nutrient solution increased the SPAD index in both spinach cultivars”.  Where is the discussion on these results?

“This is not consistent with studies that reported an excess of Fe reduced plant growth and chloro-phyll content as a consequence of oxidative stress[38]. Why is the result inconsistent? Give possible reasons!

 

Similar problems occur in most of the remaining discussion.

 

5. Conclusions (Lines 480–495)

The conclusion does not sufficiently describe the key findings of the study.

A conclusion of a research paper should be written as a home-take-away message for readers by focusing on the innovative aspects of the research.

What is the innovative finding of this research?

What are the key findings of the research and their interpretations?

How can the findings address the problems stated in the Introduction?

What are the implications of the key findings to improve indoor farming of vegetable crops and address iron deficiency in humans?

What gaps were found to bridge through future research?

What future research should be done to consolidate the findings of this research?

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

Thank you for taking the time to read our article and for your meaningful comments. Please see the attachment. 

Sincerely, Authors

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I am satisfied on authour's reply.

Author Response

We thank for Reviewer for the positive evaluation of our manuscript.

Reviewer 3 Report

 

Abstract (Lines 10–29)

 

Still there is a serious problem with this abstract:

At the start, the abstract still lacks the rationale (problem statement) of the study.

It also lacks the specific objective of the study.

In this abstract, the major results are not clearly described in quantitative terms (how much increase or how much decrease? two-fold, thee-fold, or in by how much percentage?).

The abstract also does not conclude the key results with quantified key data.

The abstract also shown no implication of the results.

 

Conclusions (Lines 495–509)

A conclusion of a research paper should be written as a home-take-away message for readers by focusing on the innovative aspects of the research.

What is the innovative finding of this research?

What are the key findings of the research and their interpretations?

How can the findings address the problems stated in the Introduction?

What are the implications of the key findings to improve indoor farming of vegetable crops and address iron deficiency in humans?

What gaps were found to bridge through future research?

What future research should be done to consolidate the findings of this research?

Author Response

We thank the reviewer for their additional feedback. With respect to the comments about the abstract, we respectfully disagree with many of the statements. Also, the abstract is limited to 200 words in this journal. Our current draft has 198 words and therefore the additional detail requested by the reviewer is not possible.

Some of the requested content is already present in the updated draft:

Reviewer: At the start, the abstract still lacks the rationale (problem statement) of the study. It also lacks the specific objective of the study.

Our response: The objective is clearly stated in the second sentence: "The aim of the study was to evaluate the effects of B (peak = 450 nm) and R (peak = 665 nm) light ratios (R:B) of 9:1, 3:1 and 1:3 on growth, metabolic response, and accumulation of mineral nutrients in spinach ‘Corvair F1’ and ‘Space F1’ grown in hydroponic solutions with different iron (Fe) concentrations (2, 5, and 15 mg L–1)."

Reviewer: In this abstract, the major results are not clearly described in quantitative terms (how much increase or how much decrease? two-fold, thee-fold, or in by how much percentage?).

Our response: There is not sufficient room to provide quantifiable information, since values depended in part on the spinach cultivar as well as the R:B and Fe concentration.

Reviewer: The abstract also shown no implication of the results.

Our response: The implication of the results is in the last sentence: "The overall nutritional quality of spinach could improve with lighting and Fe biofortification strategies"

Reviewer: [Many comments about the conclusion]

Our response: We have re-written this section with the comments in mind. We believe this draft meets many (if not all) of the suggested changes.