Zinc and Selenium Biofortification Modulates Photosynthetic Performance: A Screening of Four Brassica Microgreens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review Report

The manuscript presents a valuable investigation into the effects of zinc and selenium biofortification on Brassica microgreens, with particular focus on photosynthetic performance through chlorophyll fluorescence and pigment analysis. While the study provides interesting insights, several improvements are needed to enhance its scientific rigor and clarity. Particularly, I suggest including a comprehensive analysis of the morphometric traits across the different genotypes under the various treatments. Subsequently, It could be beneficial correlating them with the differential photosynthetic performances.

Abstract
The abstract requires restructuring to emphasize key findings:

1. Condense or remove lines 13-18 to prioritize results
2. Quantify observed variations (e.g., "Zn treatment increased pigment content by X% in genotype Y")
3. Highlight differential genotypic responses to treatments
4. Include key physiological parameters affected

Introduction
The introduction should be more focused:

1. Condense suggested paragraphs (lines 50-60, 98-119, 123-133)
2. Incorporate recent references on Brassica novel foods (doi:10.1016/j.scienta.2024.113408; doi:10.3390/foods9091226)
3. Fully specify Latin names and varieties

Materials and Methods
Methodological clarifications needed:

1. Specify seed suppliers with complete details (replace generic "local supplier")
2. Report electrical conductivity of fertigation solutions
3. Clarify growth substrate composition if not provided
4. Detail statistical analysis procedures

Results
The results section requires expansion:

1. Include comprehensive agronomic trait analysis (fresh weight, hypocotyl length, leaf area)
2. Correlate morphological changes with photosynthetic parameters
3. Label treatment concentrations clearly in Figures 1-2 (0, 2, 5, 10 mg/L Se; 0, 5, 10, 20 mg/L Zn)
4. I suggest separating results from the discussion section.

Author Response

Reviewer 1

R1: The manuscript presents a valuable investigation into the effects of zinc and selenium biofortification on Brassica microgreens, with particular focus on photosynthetic performance through chlorophyll fluorescence and pigment analysis. While the study provides interesting insights, several improvements are needed to enhance its scientific rigor and clarity. Particularly, I suggest including a comprehensive analysis of the morphometric traits across the different genotypes under the various treatments. Subsequently, it could be beneficial correlating them with the differential photosynthetic performances.

Autor`s response (AR): The Authors thank the reviewer for insightful comments that strengthened the manuscript. We have addressed each point and incorporated revisions where appropriate. Changes can be tracked in the revised manuscript (green-labeled text) and supplementary material.

Abstract
R1: The abstract requires restructuring to emphasize key findings:

1. Condense or remove lines 13-18 to prioritize results
2. Quantify observed variations (e.g., "Zn treatment increased pigment content by X% in genotype Y")
3. Highlight differential genotypic responses to treatments
4. Include key physiological parameters affected

AR: The Abstract has been rewritten (Lines 15-36) as suggested.

R1: The introduction should be more focused:

1. Condense suggested paragraphs (lines 50-60, 98-119, 123-133)
2. Incorporate recent references on Brassica novel foods (doi:10.1016/j.scienta.2024.113408; doi:10.3390/foods9091226)
3. Fully specify Latin names and varieties

AR: Thank you for your valuable suggestions. we have condensed the indicated paragraphs. The revised version presents key concepts more concisely while preserving all relevant information. Please see the lines 62-67, 111-121 and 122-135 in the revised manuscript.

As suggested, we have addressed second comment by incorporating recent findings on Brassica novel foods from the suggested references into the revised manuscript, as requested. The following can be found in lines 54-61 in the revised manuscript.

We have specified Latin names and varieties. Please see the lines 126-129 in the revised manuscript.

Materials and Methods
R1: Methodological clarifications needed: Specify seed suppliers with complete details (replace generic "local supplier").

AR: Complete details for seed supplier was specified: “…purchased from a local supplier Lokvina d.o.o. (Savska Ves, Čakovec, Croatia), certified with an ecological certificate for the providing of high-quality organic seeds.“ (lines: 142-143).

R1: Report electrical conductivity of fertigation solutions.

AR: As requested, electrical conductivity in tap water (control) and aqueous Se and Zn solutions was provided: “Electrical conductivity (EC) of solutions was measured with pH/conductivity meter (SevenCompact Duo; Mettler–Toledo GmbH, Greifensee, Switzerland) and ranged from 0.929 ± 0.008 dS/m in control (tap water) to 0.943 ± 0.005 dS/m in aqueous Se and Zn solutions, indicating that no salinity stress affected results.” (lines 151-154).

R1: Clarify growth substrate composition if not provided.

AR: As described, microgreens were grown hydroponically in tap water using floating systems in plastic containers (Lines 144/154-155) while the treatments were prepared as aqueous solutions of sodium selenate (Na2SeO4) or zinc sulfate heptahydrate (ZnSO4 x 7H2O), respectively, at different concentrations.

R1: Detail statistical analysis procedures.

AR: Statistical analysis was rewritten to include more details, including software packages. We also added Correlation analysis as suggested (lines 240-258). 

Results
R1: The results section requires expansion: Include comprehensive agronomic trait analysis (fresh weight, hypocotyl length, leaf area).

AR: A comprehensive analysis of agronomic/morphometric traits is included in new supplementary table (Table SA) and Table 1 in main text summarizing shoot (hypocotyl) length (cm), root length (cm), total plant length (cm), and dry weight proportion (DW/FW) across Brassica microgreens and treatments. Also, a paragraph (Lines 589-620 and 644-672) was added in the Results and Discussion section (green-labeled text). Since the Chl a+b/Chl data that was part of the first MS version were included in correlations, Figure 6 was removed and Chl a+b/Chl data were also added into new Table 1. The M&M section was updated accordingly (lines 190-192) as well as the Reference list.

R1: Correlate morphological changes with photosynthetic parameters

AR: New text (Lines 589-620 and 644-672) and references were added. Also, the Supplementary Tables S9 and S10 were added to correlate morphological changes to photosynthetic parameters

R1: Label treatment concentrations clearly in Figures 1-2 (0, 2, 5, 10 mg/L Se; 0, 5, 10, 20 mg/L Zn)

AR: Figures 1 and 2 were updated with clear concentration labels in Figure captions (Lines 360-361 and 378-379).

R1: I suggest separating results from the discussion section.

AR: The Authors thank the reviewer for the thoughtful suggestion to separate the Results and Discussion sections. After careful consideration, we opted to maintain an integrated format. Given the complexity of our dataset—particularly the chlorophyll fluorescence parameters and multivariate analyses such as CDA—an integrated approach allows for immediate interpretation of findings, which we believe improves clarity and readability. Separating the sections would likely lead to redundancy and fragmentation of the narrative, especially when physiological interpretations are directly tied to visual data outputs.

To address the reviewer’s concern and improve structure, we have introduced clear subheadings within the integrated Results and Discussion section to better delineate individual topics and ensure a coherent and organized flow of information.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Only one comment: taking into account the investigation of Se/Zn biofortification it is hoghly desirable ton provide the data of Se/Zn bioaccumulation levels in microgreens

minor comment:

Line 242 ‘The dose nested’- change to ‘The dose tested’

Comments for author File: Comments.pdf

Author Response

Reviewer 2:

Comments to the manuscript: Zinc and Selenium Biofortification Modulates Photosynthetic Performance: A Screening of Four Brassica Microgreens

Autor`s response (AR): The Authors thank the reviewer for insightful comments that strengthened the manuscript. We have addressed each point and incorporated revisions where appropriate. Changes can be tracked in the revised manuscript (blue-labeled and/or green-labeled text) and supplementary material.

Major Comments:

R2: Please, indicate the levels of Se/Zn accumulation by microgreens: they should differ among the species chosen. May these differences (if any) be connected with the peculiarities of photosynthesis changes?

AR: Thank you for your valuable suggestions. Since element accumulation is central to biofortification strategy, we have added preliminary results of Se/Zn uptake as a single measurement (Figure 6). This study focused primarily on photosynthetic performance rather than direct quantification of Se/Zn uptake, as the goal was to screen for physiological adaptations. However, since species-specific differences could influence electron transport efficiency, future studies will include uptake values for a detailed analysis of species-specific responses. Literature indicates variability in Se and Zn accumulation in different Brassicaceae. For example, higher Se accumulation may enhance PSI efficiency via its antioxidant role, while excess Zn might over-reduce the Q_A pool, impairing performance. We have added this in the Results and Discussion section (Lines 674-722). The M&M (lines 194-204) and Reference list was updated accordingly

R2: Line 93 Especially, Zn application requires careful optimization due to its dual role as both an essential micronutrient and a potential heavy metal pollutant’- think, it is not ‘especially’ as the same pattern is typical for Se possessing a very narrow range of safe doses for plants. In general, both Se and Zn along with all other elements may affect plant according to the well-known law of optimum when any factor of the environment demonstrates a beneficial effect on living organism at certain dose: both higher and lower levels may lead to a harmful effect (the so-called Shelford’s law of tolerance). In this respect high doses of Se may also cause oxidative stress.

AR: We fully agree with the reviewer’s observation. In response, we have revised the paragraph to reflect a more general perspective, emphasizing that this pattern applies not only to Zn but also to Se and other micronutrients. Please see the lines 105-110 in the revised manuscript.

R2: I propose to indicate in the Introduction section that Brassicaceae plants demonstrate high prospects of Se biofortification due to high content of sulfur (chemical analog of Se) which is easily substituted by Se.

AR: We appreciate the reviewer’s valuable suggestion. As proposed, we have revised the Introduction to include a text highlighting the high potential of Brassicaceae plants for Se biofortification due to their naturally high sulfur content. Please see the lines 95-102 in the revised manuscript.

R2: What was the size of microgreen used in the investigation? Provide either microgreen length or/and a photo. What were the differences between 4 species?

AR: A comprehensive analysis of agronomic/morphometric traits is included in new Table 1 in main text and supplementary table (Table S9) summarizing shoot (hypocotyl) length (cm), root length (cm), total plant length (cm), and dry weight proportion (DW/FW) across Brassica microgreens and treatments. Also, a paragraph (Lines 586-620 and 644-672) was added in the Results and Discussion section (green-labeled new text). Since the Chl a+b/Chl data were included in correlations, that were part of the first MS version, Figure 6 was removed and Chl a+b/Chl data were added into new Table 1. The M&M section was updated accordingly (lines 190-192) as well as the Reference list.

R2: Explain the reason of using such concentrations of Se and Zn

AR: Concentrations were selected based on the extensive literature review to achieve biofortification without causing toxicity. First, selenate over selenite was chosen since it was shown to have positive effect on biomass production and better translocations to upper parts of the plants (Ramos et al., 2010; Zafeiriou et al., 2022). Further, Se (2-10 mg/L) was chosen because low doses enhance antioxidant status without severe growth inhibition (Mezeyova et al., 2022; Tavan et al., 2024; Hossain et al. 2021), with higher concentrations serving as tolerance thresholds. Zn (5-20 mg/L) was chosen since it was determined that 5-10 mg/L increases Zn content by up to 281% (Di Gioia et al, 2019; Poudel et al., 2023), with 20 mg/L as the upper limit. Therefore, named references were cited and additional sentence was added to Methods: (Lines 149-150): “Doses were chosen from the literature review to balance nutritional enhancement and physiological safety (25, 42-47)”.

R2: Taking into account that both Se and Zn participate in antioxidant defense it seems highly important to evaluate microgreen antioxidant status.

AR: We agree with the reviewer that assessing antioxidant status would provide valuable complementary insight, especially given the known roles of Se and Zn in redox regulation. However, the scope of our study was intentionally focused on photosynthetic performance metrics as a rapid, non-destructive screening approach. To acknowledge this important aspect, we have now included the following sentence in the Conclusion section (Line XXX): “Future research should focus on analyses of antioxidant status to better link Se and Zn accumulation with oxidative stress defense mechanisms, which may help explain the observed species-specific responses.”

Minor comments:

R2: Decipher PI and DF in the footnote of Figure 4. The same for MR in Figure 5.

AR: Figure 4 caption now includes: PI_tot (performance index) and DF (driving forces); Figure 5 MR (Modulated reflection at 820 nm).

R2: Line 242 ‘The dose nested’- change to ‘The dose tested’

AR: The unintentional error was corrected.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors addressed all the reviewer's comments

Author Response

We thank the reviewer for their constructive feedback and valuable suggestions that improved the clarity and quality of the work.

Kind regards

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors. Please add the  chemical forms of Se and Zn used in the investigation at the end of the Introduction section while describing the aim of the work and especially in the Abstract . Keywords should also contain Se/Zn supplementation/biofortification 

Author Response

R2: Dear authors. Please add the chemical forms of Se and Zn used in the investigation at the end of the Introduction section while describing the aim of the work and especially in the Abstract. Keywords should also contain Se/Zn supplementation/biofortification.

AR: We appreciate the reviewer’s valuable suggestion. As proposed, we have added (red lebeled text) chemical forms of Se an Zn in Abstract (line 20) and Introduction (line 127), as well as incorporated Se/Zn supplementation/biofortification in the keywords (line 37).