Review Reports - Influence of the Sunlike Light Spectral Composition on Radish in Controlled Environment Agriculture: Morphophysiological Characteristics and Diffuse Reflection Indices of Leaves

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study concerns an actual topic and an important but less studied crop in indoor and vertical farming systems. The manuscript is well and clearly organized and a fluent and clear English is used. I apologize for the length of my revision report, it is to be as much clear as possible in my comments and suggestions. The most important comments refer to the interpretation of results. I wrote several examples and respective explanations to support the doubts and to suggest corrections and improvements. I hope the authors will find them useful and will not be annoyed by the number of comments. It is for the best of their valuable article.

Please, find the comments in the attached file.

Best regards

Comments for author File: Comments.pdf

Author Response

RESPONSE to the Reviewer 1.

Dear Reviewer,

We sincerely appreciate your clear, detailed and friendly review. We hope that the work we have done has significantly improved the quality of the presentation of the results in the manuscript. Many of your questions and comments were very useful to us for our future work. If there are any aspects of the revised version that do not meet your expectations, we are willing to continue working with you to improve the manuscript until it meets the high standards expected by MDPI.

Kind regards,

The authors.

To the authors,

In the abstract, even if it is said that this research is important because "Creating an optimal light environment is crucial for achieving high yields in controlled environment agriculture (CEA) conditions", I highly recommend to shortly define the novelty and originality of this study. This because the comparison with HPS and LED lamps has been largely investigated for several years up to date, and HPS lamps are poorly chosen compared to LED lamps in indoor cultivation systems and vertical farming, to which the authors focus within the CEA systems.

In the abstract, considering that you studied two very different varieties, a short comment on the effects on this aspect should be added (e.g. presence or lack of variety-dependent effect).

Response 1: We greatly appreciate the reviewer's valuable suggestion. We have adjusted the abstract in response to the comments of our reviewers:

Lines 15-33:

Creating an optimal light environment for different crops is crucial for achieving high yields under controlled environment agriculture conditions. Currently, there are no optimal technologies, including lighting technologies, for growing root crops (in particular radish) in CEA. This study examined the effects of HPS and three original sunlike full-spectrum LED lamps on the morphophysiological characteristics and the diffuse reflectance indices of leaves of two contrast radish cultivars. It was found that a higher blue light content (24%) in the spectrum of the LED 3 lamp contributed to the formation of radish plants with a more compact leaf rosette and maximum yield of roots (up to 19%) compared to the other two types of LED lamps. When treated with LED 3, photosynthesis efficiency was probably higher compared to LED 1 and LED 2, which led to a significant decrease in reflected radiation, especially in the blue and red ranges (by 5-143% and 32-86%, respectively). It was found that the genotype had a significant effect on all mor-phophysiological parameters of radish, while lightning treatment only affected the inte-gral parameters (Pr – proportion of root crop, and Ai – attraction index) and leaf thickness. However, lightning treatment exhibited a greater impact on leaf reflection indices compared to genotype, especially those related to chlorophyll content. The results of the study indicate that LED 3 lamps, simulating natural light at midday, are suitable for production of radish root crops under CEA conditions, since the mass of root crops and yield per square meter did not differ from those of under HPS reference treatment.

Paragraph 77-88 References should be added.

Response 2: Thank you for your suggestion. Links have been added and the text has been updated:

Lines 77-109:

Optical methods of non-invasive control, which make it possible to evaluate the growth and the health of plants and their response to the applied treatments in the genotype-environment system, can serve as an effective tool for optimizing technologies for growing various crops in CEA. The optical properties of leaves are primarily determined by the photosynthetic pigments in their tissues (chlorophyll a, b, and carotenoids), but also depend on leaf structure, the presence and concentration of non-photosynthetic com-pounds (anthocyanins, flavonols), and water. These properties are a key characteristic of plants, which makes it possible to estimate the amount of photosynthetically active radiation absorbed, the intensity of photosynthesis and, ultimately, the physiological state, as well as predict crop yields [14-16].

Qualitative and quantitative changes in the pigment content of plant leaves inevitably lead to changes in their optical properties [15-17]. This is the basis for understanding the mechanisms of plant physiological reactions, resistance, and adaptation, which are essential for developing effective crop-growing technologies and improving methods for rapid phenotyping. To interpret spectral measurements, several reflectance indices have been developed. These indices, in contrast to simple differences between two reflection values at different wavelengths, are normalized values that more accurately characterize leaf reflectivity [18-22]. The authors who developed the formulas for normalized reflectance indices tested them on various plant species and compared with the results of bio-chemical assessment of chlorophyll - ChlRI [18], carotenoids - SIPI [19], anthocyanins - ARI [21,22] amount in leaves. Also, the relationship between the photochemical reflectance index – PRI and the epoxidation of pigments of the xanthophyll cycle, the efficiency of light use and the efficiency of photosynthesis were determined [19]. We have previously used these indices both in field studies and in the CEA for phenotyping and evaluating the dynamics of changes in biomass, pigment composition, and yield prediction of wheat and lettuce plants that are deficient in mineral nutrients and water [14-16,23].

In response to many factors, the spectral and optical characteristics of radiation reflected from the leaf surface change even before external symptoms appear on the plant itself [17]. It has been demonstrated that the reflectance indices can be used to evaluate the impact of lighting quality on plant development [24].

In the whole text, when referring to a cultivar, usually or the abbreviation "cv." or the single inverted commas are used, not both at the same time.

Response 3: Thank you very much for your suggestion. We have fixed the reference to a cultivar, now it is cv. Pernot (cv. P), or cv. Peterburgskiy fioletovyy (cv. Pf).

Figure 1C. Showing the HPS treatment along with the photos of the three LED treatments would complete the representation, especially because HPS is the control.

Response 4: We greatly appreciate the reviewer's valuable suggestion. Unfortunately, we did not take good photos of radishes under the HPS lamps. We have adjusted Figure 1 and divided it into two figures: Figure 1 and Figure 2:

Figure 1. Radish plants used in the experiment. A – cv. Pernot, B – cv. Peterburgskiy fioletovyy.

Figure 2. Radish under different LED lamps. A – LED 1, B – LED 2, C – LED 3.

Line 135 for the number of leaves, the rational of considering the leaf in the count could be added. To give an example, leaves longer than X mm were considered.

Response 5: Thank you very much for your suggestion. We added the rational of considering the leaf in the count:

Lines 153-156:

During the harvest, the main morphophysiological parameters were measured: the fresh mass of the root crop (Rm, g) and leaves (Lm, g), the number of leaves (Nl, pcs.); the number of developed leaves with a leaf blade length of more than 8 cm was taken into account when we calculated an area of radish leaves.

Line 144-145 Ei index: I understand that you called it "economic efficiency index" because it refers to the mass economic interest of the plant. However, considering the formula referring to biomass, it is basically a harvest index. The reference to "economic" efficiency, it gives the expectation of an economic calculation, referring to money, and not just to the biomass harvest.

Response 6: Thank you very much for your suggestion. We agree with your comment and have made changes:

Based on the data obtained, the following selection indices were calculated, characterizing the yield:

Lines 153-156:

Pr – the proportion of root crop:

Pr = Mass of the root / Mass of the whole plant

Line 150. A clarification on the experimental layout is required. Two consecutive experiments were performed? How many repetitions per treatment per experiment? In the current form I understand that you repeated the experiment in the time twice but without replicates. Please, clarify in the manuscript.

Response 7: We greatly appreciate the reviewer's valuable suggestion. We have clarified in the manuscript:

Lines 171-174:

The experiment was repeated twice. In each experiment, the number of plants evaluated was 8; since similar average values of the studied parameters were obtained in both experiments, statistical processing was performed for a combined sample of 16 radish plants.

In case you had only two overall replicates of the treatments, this is usually considered not enough to properly support the statistics as normally not less than three replicates are used. Line 162 "(Table 1, Figure 2)". the reference to the table and figure could be moved at line 158 just before the two points.

Response 8: Thank you very much for your suggestion. As we conducted the experiment in controlled conditions with identical environmental factors, we believe this repetition will be sufficient to obtain reliable results. In fact, three experiments were conducted, but we consider the first one to be "tentative"; although the results were similar to the next two, the repetition (number of plants) was lower. We did not use the data from the first experiment when writing the manuscript.

Section 2.2, A small suggestion on Table 1 and related comments in paragraph 173-184. I see the point of compering the light spectra of lamps; however, I am not fully convinced of the choice of commenting the statistical differences in the material and methods and in a position where you have not presented yet the statistical analysis section. Maybe it would be better to have the synthetic plots or a schematic description of the lamps and this table somewhere else (results or supplementary material, e.g.). For example, instead of giving only the К of the lamps in lines 160-162, you could add there also the spectral ratios (meaning the values you have in the plots of figure 2), like R:G:B:... X:Y:Z:...

Response 9: Thank you very much for your suggestion. We added the spectral ratios (R:G:B):

Lines 178-188:

After sowing, seedling trays with seeds were placed under specific lighting conditions. Four lighting treatments were used in the study (Table 1, Figure 2). HPS (OOO Re-flax, Moscow, Russia), which we had previously used for breeding of the cv. Peterburgskiy fioletovyy, were used as a reference. HPS red : green : blue ratio (R:G:B) is 8,5 : 9 : 1 (to ease to count and understand, red is taken as 600-700 nm, green is taken as 500-600 nm and blue is taken as 500-600 nm; and the blue light content is taken as 1).

Three original full-spectrum LED lamps (Figure 2A-C) were developed and manu-factured at the Agrophysical research institute (AFI):

1) LED 1 with a color temperature of 3000 K, R:G:B is 5,2 : 3,3 : 1;

2) LED 2 with a color temperature of 4000 K, R:G:B is 2,3 : 2 : 1;

3) LED 3 with a color temperature of 5000 K, R:G:B is 1,5 : 1,5 : 1.

Line 195-196, please add when the spectra of reflected radiation from leaf surfaces were recorded, e.g., X days from sowing.

Response 10: Thank you very much for your suggestion. We added information:

Lines 239-244:

The measurements were carried out on the 20th day after sowing, 5 days before harvesting. The diffuse reflectance spectrum of the leaf is displayed on the computer screen as a percentage of the reference source's reflectance and can be saved as a digital file. The resulting leaf reflectance spectra were used to calculate reflectance indices, which are closely related to the activity of the photosynthetic apparatus and the physiological state of plants.

Line 220-221. Please add "nm" also to 799.1 and 551.8.

Response 11: Thank you very much for your suggestion. We added nm:

Line 265-266

R_NIR = 799.1 nm, R_gr = 551.8 nm, and R_re = 703.4 nm

Section 2.3.2 there are quite discursive explanations that looks like more appropriate in an introduction to the use and the choice of these indices. Particularly, lines 246-252 about heterogeneity in PRI could fit a discussion section rather than Materials and Methods.

Response 12: Thank you very much for your suggestion. We have partially transferred information about the PRI index to the introduction and removed excessive explanations about heterogeneity in PRI:

Line 97-99

Also, the relationship between the photochemical reflectance index – PRI and the epoxidation of pigments of the xanthophyll cycle, the efficiency of light use and the efficiency of photosynthesis were determined [19].

1 suppose it should be called "Results and Discussion" and not just "Results".

Response 13: Thank you very much for your suggestion. In the new version of the manuscript, Section 3 is called "Results and Discussion" (Line 311).

Lines 269-273. Please add references to the tables/figures where the reader can check the info. Lines 279-282 repeat the concept, so I would suggest making these lines concise.

Response 14: We greatly appreciate the reviewer's valuable suggestion. We have significantly rewritten the section 3.1, applied a new method of statistical data processing and description, and removed unnecessary repetitions:

Lines 323-325:

Genetically determined differences between the cultivars were generally found in all studied parameters, except for LA (Figures 4A-E, Table S1). Higher root mass in all treatments for cv. Pf can be explained by transgressive breeding [6].

Figure 3 caption. It is useful to the reader the explanation in the caption of the abbreviations in the figures. For example: A- root mass (Rm); В - leaves mass (Lm), etc...

Response 15: Thank you very much for your suggestion. The latest version of the manuscript presents a modified (after additional statistical processing) version of this figure, now it has the number 4. We've deciphered all the abbreviations in the caption:

Lines 338-344:

Figure 4. Effects of crop variety (Pernot (P) and Peterburgskiy fioletovyy (Pf)) and spectral lighting treatment (HPS, LED 1, LED 2, LED 3) on growth and biomass parameters. Data are presented as mean ± standard error. For each subfigure, uppercase letters denote sig-nificant differences between cultivars, and lowercase letters denote significant differences among lighting treatments (Tukey's HSD test, p<0.05). (A) Root mass (g). (B) Leaf mass (g). (C) Proportion of root crop (Pr). (D) Attraction index (Ai). (E) Number of leaves. (F) Leaf thickness (µm).

About 3. Please, apologize for the long text, is to be as clear as possible. The current representation with letters referring to all the single combinations of light*genotype is misleading to the reader. Checking table S1, the interaction is significant only in Root shape index, so why presenting the posthoc letters of the interaction for all the parameters? To check the significance of main effects and interactions, the reader is obliged to check the p value in supplementary table, which are "supplementary" but essential for the understanding of the results.

Response 16: We greatly appreciate the reviewer's valuable suggestion. We conducted an additional statistical analysis of the data obtained, including post-hoc analysis. The revised version of the manuscript includes all new statistical results, Figure 3 (now Figure 4), as well as tables 3-5, now contain letters that reflect genetic and environmental differences (Different letters in the same reflectivity index indicate significant differences between treatments: uppercase letters denote significant differences between cultivars, and lowercase letters denote significant differences among lighting treatments (Tukey's HSD test, p < 0.05)). Table S1 has also been rewritten, in particular, all the characters that are not mentioned in the text of the manuscript have been removed from there. Accordingly, we have rewritten the text of sections 3.1 and 3.2 in accordance with the obtained values.

Discussion in lines 373 to 411 could be more concise on the general description of spectrum-plant relation.

Response 17: Thank you very much for your suggestion. The discussion on lines 373-411 (in revised version – on lines410-411) has become shorter, and all this part of manuscript has been rewritten:

Lines 411-427:

Blue and red light play a key role in photosynthesis, although their effects on plants can be different. Many publications have demonstrated the stimulating effect of red light on the height, diameter, and aboveground biomass of various crops, in particular, lettuce [30-32], pak choi [33], and tomato [34]. In our study, the maximum values of the aboveground part (leaf mass) were also found in the LED 1, which is characterized by the maximum (43%) proportion of red light in the spectrum (Figure 3,4B). Similarly, the presence of blue light is necessary for normal plant growth, and the addition of blue wavelengths in certain pro-portions (optimal for a specific crop or variety) increases yield and improves quality for a number of crops in CEA [35-38]. At the same time, blue light can also reduce plant growth. High proportions of blue light have been shown to reduce the biomass of the above-ground part of lettuce [39-41], the height and biomass of cucumber shoots [41], and the dry weight of cucumber, pepper, and tomato shoots [42]. Additionally, blue light can stimulate the development of root systems and increase root biomass in various crops [42-45]. Our experiments also showed some inhibition of the growth of the leaves and, simultaneously, stimulation of the growth of the root crop in both radish cultivars under LED 3 treatment, which has the highest proportion of blue light in its spectrum (Figures 3,4A).

Lines 458-459 “It was found that the leaves of the cv. ‘Pf’ reflected less PAR radiation and apparently absorbed more”. Where can the reader find this result? If referred to table 3,1 do not see letters of statistic supporting this affirmation.

Response 18: Thank you very much for your suggestion. The letters of statistic supporting were added, and the results were revised:

Lines 479-427:

Significant differences were observed in the amount of radiation reflected from the leaf surface across all spectral ranges, both between the lamps and between the studied cultivars. It was found that the leaves of the cv. Pf reflected less PAR radiation and apparently absorbed more, suggesting more intensive growth and biomass accumulation com-pared to the cv. P. So, under HPS treatment, the amount of PAR radiation reflected from the leaf surface were 28.8% (cv. Pf) and 40.1% (cv. P); under LED 1 – 46.1% and 55.2%, respectively; under LED 2 – 37.3% and 43.3%; and under LED 3 – 29.9% and 39.7% for the cvs. Pf and P, respectively.

Lines 488-500.1 suggest shortening on this explanation since result and discussion section is already long and this is mainly an explanation on why the authors used the method.

Response 19: Thank you very much for your suggestion. We removed unnecessary information.

Lines 504-506. Where can the reader find the results referred to this statement about “patterns of change depending on the PAR spectral composition during the growing season”? I understood that the spectrum was fixed through the growing cycle of the present experiment Data or analysis on variation across the growing season are not reported in results. Please clarify in the manuscript if this sentence refers to a missing citation to another research.

Response 20: Thank you very much for your valuable suggestion. You are absolutely right, this is an incorrect expression, we measured the spectra only once. The expression " during the growing season" has been removed.

Lines 506-507, 514-515. “The smallest differences in this indicator were found between the HPS and AFI-3, and the largest between the HPS and AFI-1”. Same as above, the letters of statistical analysis are mainly the shared, indicating no significant differences.

Response 21: Thank you very much for your valuable suggestion. The letters of statistical analysis are added now, and significant differences are indicated.

Table 5. Please add in caption or foot notes the full names of the abbreviation used as reflectivity index column.

Response 22: Thank you very much for your suggestion. The caption was added:

Lines 558-564:

Note: Data are presented as mean ± standard error (M±SEM). Different letters in the same reflectivity index indicate significant differences between treatments: uppercase letters denote significant differences between cultivars, and lowercase letters denote significant differences among lighting treatments (Tukey's HSD test, p < 0.05). SIPI – leaf struc-ture-independent pigment index; ARI-1 and ARI-2 – anthocyanin reflectance indices; PRI - photochemical reflectance index; R800 - light scattering inside leaf tissues. Cv. Pf – cultivar Peterburgskiy fioletovyy, cv. P – cultivar Perno.

On the base of the doubtful result interpretation, I kindly suggest reviewing the conclusions, to make them consistent with the results statistically significant.

Response 23: Thank you very much for your valuable suggestion. We have corrected the text in the manuscript in those places where it concerns the reliability of statistical differences, and also corrected the conclusion. We believe that despite the fact that the differences between the tritements in terms of root crop weight and yield were not statistically significant, the trend we identified associated with an increase in these indicators with an increase in the proportion of blue light in the spectrum of the LED lamp is quite reliable. Therefore, we consider it possible to write about this (as a trand), especially since the reflection indices also confirm our conclusions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Experiment is well-designed with clear presentation of results relevant for CEA lighting optimization. However, novelty is moderate, methods are technically sound but not innovative enough for novelty expectations, unless reframed and english requires substantial editing. Interpretation of results are sometimes overstated relative to statistics and some methods lack essential detail for reproducibility which is accompanied that some statements do not fully align with Table S1 ANOVA results.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English requires substantial editing, especially for:

“Light source” vs. “lighting environment” inconsistently used.
Units should follow MDPI standards: μmol m⁻² s⁻¹, not μmol / (m2 × s).
Figures need clearer legends (symbols for cultivars, meaning of letters).
Equation formatting needs LaTeX or clearer structure.
Reference formatting inconsistent.
Table S1 check and write in the manuscript consistently.

Author Response

RESPONSE to the Reviewer 2.

Dear Reviewer,

We sincerely appreciate your clear and detailed review. We hope that the work we have done has significantly improved the quality of the presentation of the results in the manuscript. Many of your questions and comments were very useful to us for our future work. If there are any aspects of the revised version that do not meet your expectations, we are willing to continue working with you to improve the manuscript until it meets the high standards expected by MDPI.

Kind regards,

The authors.

Suggestions for improvement:

Emphasize custom engineering of AFI LED modules. Emphasize linking reflectance indices with yield prediction. To do not highlight “mechanistic” claims unless statistically supported.

Response 1: We greatly appreciate the reviewer's valuable suggestion. In order to improve the presentation of the results, novelty and originality, we have added the following statements:

Lines 27-31: It was found that the genotype had a significant effect on all morphophysiological parameters of radish, while lightning treatment only affected the integral parameters (Pr – pro-portion of root crop, and Ai – attraction index) and leaf thickness. However, lightning treatment exhibited a greater impact on leaf reflection indices compared to genotype, especially those related to chlorophyll content.

Lines 74-76: In this regard, it is important to develop LED modules with a full spectrum of radiation and select optimal spectra for growing various types of crops, in particular radish, taking into account the interaction between the genotype and the environment.

Lines 191-201: When creating full-spectrum LED lamps, we used a new generation of LEDs that emit purple light and are coated with a three-component phosphor that converts the light into a PAR with full red, green, and blue components. The novelty of the developed LED lamps lies in the formation of full-spectrum radiation simulating solar illumination at different times of the day to identify the most natural, functional conditions to which plants are evolutionarily more adapted. Unlike conventional red-blue and white LED phytolamps, the spectrum of original LED lamps includes all wavelengths. For example, red-blue lamps completely lack the green region and white lamps exhibit characteristic dips in the 480-520 nm and 600-640 nm ranges. Furthermore, the use of diffuse secondary optics with high light transmittance for our LEDs creates the high isotropic light field to create uniform growing conditions.

Lines 538-541: Correlation analysis of the relationship between the chlorophyll index values, plant morphometric characteristics, and yield of two radish genotypes revealed a close relationship (r² = 0.50-0.62, p < 0.05) between ChlRI (as well as Chl_gr) with the leaf assimilating surface area (LA) and leaf thickness (Lt).

Lines 621-626: According to the results of our study, carried out on two radish cultivars grown under HPS and LED lamps, R₈₀₀ is negatively linearly related to parameters characterizing the productivity of the photosynthetic apparatus - LA, Nl, and Lm:

LA = 549.64 - (3.213 × R₈₀₀); r = -0.725; p = 0.042; r² = 0.525

Nl = 9.189 - (0.047 × R₈₀₀); r = -0.811; p = 0.015; r² = 0.658

Lm = 18.69 - (0.155 × R₈₀₀); r = -0.804; p = 0.016; r² = 0.647

1. Methods: Reproducibility Needs Improvement Several key methodological details are missing: LED fixture specifications: spectral distribution as actual SPD curves (only relative proportions given) LED manufacturer/power consumption, distance from lamp to canopy, uniformity of light distribution (PAR mapping).

Response 2: Thank you for your suggestion. LED1-LED3 lamps were developed as experimental light modules in AFI, and their effectiveness has been studied on various crops, with radish taking an important place among them. Radish cultivation techniques at CEA have not yet been developed, but we believe that our research will help improve our understanding of this crop's requirements for light spectral composition. We added Figure S1 and S2 to make better understanding of lightning treatments applied and added following statements:

Lines 139-148: Irradiance was maintained the same for all treatments – 75±5 W m-² in the PAR region. Irradiance was measured at the plant level. The distance from the lamps to the upper leaves of the plants during the experiment was 40-45 cm. The characteristics of light sources and the uniformity of light distribution over the entire irradiated surface with plants was measured using a PG200N Spectral PAR Meter (UP-Rtek, Taiwan) and did not exceed a deviation of 12% of the total illumination. To offset the effect of the differences of light distribution, the trays with plants were rotated on 90° every three days. As the plants grew, the illumination was adjusted by dimming the lights (LED 1- LED 3) or raising the light blocks (HPS). Light and temperature conditions were equalized using mirror screens around the perimeter of each lightning treatment and air conditioning.

Lines 184-188: Three original full-spectrum LED lamps (Figure 2A-C) were developed and manufactured at the Agrophysical research institute (AFI):

1) LED 1 with a color temperature of 3000 K, R:G:B is 5,2 : 3,3 : 1;

2) LED 2 with a color temperature of 4000 K, R:G:B is 2,3 : 2 : 1;

3) LED 3 with a color temperature of 5000 K, R:G:B is 1,5 : 1,5 : 1.

State whether repetitions are biological or technical, as it is unclear whether statistics is used pooled or independent replicates.

Response 3: Thank you for your suggestion. We added Lines 171-174:

Leaf reflectance measurement needs more detail: contact angle and probe distance, calibration procedure, number of measurements per plant vs. per treatment, whether spectra were averaged before indices calculation.

Response 4: Thank you for your suggestion. We added Lines 229-244:

The spectra of reflected radiation from leaf surfaces were recorded non-invasively using a fiber optic system (Ocean Optics, Largo FL, USA). This system consists of four main components: an HR2000 spectrometer, specialized SpectraSuite software, a reference tungsten-halogen light source (LS-1), and a reflection probe (R-200-7). The reflection probe has a tightly packed arrangement of six illumination fibers around a single read fiber. System provides an optical resolution of 0.065 nm in the range from 400 to 1100 nm. Be-fore leaf measurements, the reflectance spectrum of the reference source (WS-1), made of "spectralon," a material that reflects over 99% of incident radiation in the measured wave-length range was recorded. To record the spectra, the sensor was positioned in the mid-section of midlayer leaves, avoiding the central vein. On average, at least 15 spectra were recorded for each treatment. The measurements were carried out on the 20th day af-ter sowing, 5 days before harvesting. The diffuse reflectance spectrum of the leaf is dis-played on the computer screen as a percentage of the reference source's reflectance and can be saved as a digital file. The resulting leaf reflectance spectra were used to calculate reflectance indices, which are closely related to the activity of the photosynthetic appa-ratus and the physiological state of plants [16].

2. Overinterpretation in Discussion (Not supported by statistics)

Response 5: We greatly appreciate the reviewer's valuable suggestion. We conducted an additional statistical analysis of the data obtained, including post-hoc analysis:

Lines 257-265: 2.4. Statistical Analysis

Statistical evaluation of the obtained data was performed by calculating the main descriptive characteristics: mean, and standard error of the mean (SEM). Duncan's multiple-range test was used to identify differences between mean values for each characteristic. A p value of < 0.05 (5% error probability) was considered an acceptable limit of statis-tical significance. Data analysis was performed using Microsoft Office Excel 2019 and Statistica v. 13.3 (StatSoft Inc., Tulsa, OK, USA). A two-way analysis of variance (ANOVA) of morphological traits was conducted to identify significant relationships between genotype, environmental conditions (light regime), and interfactorial interactions.

The revised version of the manuscript includes all the new statistical results, Figure 3 (in revised version - Figure 4), as well as tables 3-5, now contain letters that reflect genetic and environmental differences (Different letters in the same reflectivity index indicate significant differences between treatments: uppercase letters denote significant differences between cultivars, and lowercase letters denote significant differences among lighting treatments (Tukey's HSD test, p < 0.05)). Table S1 has also been rewritten, in particular, all the characters that are not mentioned in the text of the manuscript have been removed from there. Accordingly, we have rewritten the text of sections 3.1 and 3.2 in accordance with the obtained values.

The manuscript attributes high PRI under LED light to “down-regulation” and midday depression analogies. This conclusion is speculative without photosynthetic gas exchange data. If possible include this data, or omit this in discussion.

Response 6: Thank you for your suggestion. You are certainly right; the exact answer can only be given by a parallel study of photosynthetic gas exchange. We plan to use this method in the continuation of research. We have corrected this statement:

Lines 257-265:

Lines 641-645: The observed changes in the optical characteristics of radish leaves under LED treat-ments (a decrease in chlorophyll content – ChlRI and interleaf light scattering – R800, as well as the accumulation of anthocyanins – ARI-1 and an increase in thermal dissipation – PRI) compared to HPS lamps are likely a manifestation of the "down-regulation" of photosynthetic processes, but this assumption requires a comprehensive additional study.

“Efficiency of photosynthetic apparatus is higher in AFI-3”. This is overstated. Indicators (reflectance indices) suggest changes, but do not directly quantify efficiency.

Response 7: Thank you for your suggestion. We partially agree with the remark. There is no quantitative assessment of the change in the value of the indices, but the direction of the changes and the result (correlations with yield) suggest that they are one of the symptoms of the suppression of the photosynthetic apparatus.

To strengthen novelty, you should: emphasise that radish is understudied in reflectance-spectroscopy context; clearly state why full-spectrum LEDs require assessment across genotypes and explain how the AFI LEDs differ from commercial white LEDs.

Response 8: We greatly appreciate the reviewer's valuable suggestion. In order to improve the novelty and originality, we have added the following statements:

Lines 47-54: Radish (Raphanus sativus L.) is widespread among agricultural crops and is grown everywhere [5]. Small radish is promising for production under light culture conditions, as it has all the necessary characteristics required for crops in CEA: a short growing sea-son, compact habitus and high nutritional properties, which are characteristic not only of root crops, but also, to a greater extent, of leaves. Despite the high potential of small radish for production in the CEA, this crop has been relatively poorly studied both in terms of the reaction of various samples to the light environment and in the reflection-spectroscopy context.

Also, several manuscript statements contradict or do not cite the ANOVA results, such as “light environment significantly influenced leaf area” (p=0.018), so this claim should explicitly cite Table SI., “there were no significant differences in leaf mass” is correct (p=0.607), but manuscript sometimes describes trends without stating non-significance, or “root mass increased toward AFI-3” should be soflty described.

Response 9: Thank you for your suggestion. We conducted an additional statistical analysis of the data obtained, including post-hoc analysis, as discussed above. The revised version of the manuscript includes all the new statistical results, Figure 3 (in revised version - Figure 4), as well as tables 3-5, now contain letters that reflect genetic and environmental differences (Different letters in the same reflectivity index indicate significant differences between treatments: uppercase letters denote significant differences between cultivars, and lowercase letters denote significant differences among lighting treatments (Tukey's HSD test, p < 0.05)). Table S1 has also been rewritten, in particular, all the characters that are not mentioned in the text of the manuscript have been removed. Accordingly, we have rewritten the text of sections 3.1 and 3.2 in accordance with the obtained values.

Minor Issues (English, Formatting, Clarity)

English grammar requires extensive editing.

“Light source” vs. “lighting environment” inconsistently used.
Units should follow MDPI standards: pmol m-2 s-1, not pmol / (m2 x s).
Figures need clearer legends (symbols for cultivars, meaning of letters).
Equation formatting needs LaTeX or clearer structure.
Reference formatting inconsistent.
Table SI check and write in the manuscript consistently.

Response 10: We greatly appreciate the reviewer's valuable suggestion. We have tried to significantly improve the presentation of the results: use the correct terms, the correct format for units of measurement, updated all captions to figures and tables, corrected reference formatting, edited and improved the English language (terms and grammar). Table S1 has also been rewritten, in particular, all the characters that are not mentioned in the text of the manuscript have been removed from there and from Section 2 and 3.

Suggestions for improvement:

Genotype has large effect on root mass, leaf mass, leaf number. Light environment significantly affects Ei, Ai, leaf area, leaf thickness. Interaction effects mostly non-significant except root shape index. Root length significance (p=0.631) never mentioned. Rosette height and diameter also not discussed. “higher root mass under AFI-3” is not statistically significant. “root diameter significantly affected by light” is correct but not discussed in main text.

“light significantly affects leaf number” is correct, but manuscript incorrectly states “no significant. «differences in leaf number were found”

Response 11: We greatly appreciate the reviewer's valuable suggestion. We have taken into account all your comments and tried to correct them in the text of the manuscript. The post-hoc analysis significantly simplified the understanding of the results. We hope that there are no contradictory statements left in the manuscript that are not confirmed by the results.

Required for acceptance:

Strengthen methodological description, correct statistical interpretations (especially Table SI inconsistencies), remove speculative physiological explanations, improve English style and scientific clarity, emphasize the novelty more clearly, improve figures/legends, rephrase all claims of “significant effect” only when supported by ANOVA

If these revisions are made, the manuscript should be suitable for publication.

Response 12: We greatly appreciate the reviewer's valuable suggestion. We hope that we have made these revisions, for this the manuscript has been almost completely revised and rewritten.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the effects of different full-spectrum LED lights (3000K, 4000K, 5000K) versus HPS on radish morphophysiology and leaf reflectance. The topic is timely and relevant to controlled-environment agriculture and lighting optimization.The study is well-structured and includes detailed spectral and physiological analysis. However, revisions are needed to improve scientific clarity, data interpretation, and organization.

The introduction is lengthy and mixes general CEA background with study-specific gaps.Clearly state the knowledge gap and novelty in last paragraph of introduction.
Add a sentence clearly stating why studying sun-like LEDs simulating midday solar spectrum is novel and how it advances radish lighting optimization.

PPFD is controlled (good), but light intensity uniformity measurements across canopy not reported.

Indicate whether light maps or measurements at multiple canopy points were taken.Provide PPFD uniformity details (e.g., min-max range, coefficient of variation).

Clarify whether analyses used n = 16 per treatment or n = 8 averaged over repetitions.

Many figures are crowded, difficult to read, and not self-explanatory.Increase font size,Use consistent labeling, Include significance letters more clearly, Define abbreviations in captions (Ei, AI, LA, etc.)

The manuscript suggests: Higher anthocyanins and PRI = photoprotection due to blue-rich light However: No pigment or chlorophyll lab validation was performed to confirm remote sensing indices.

The discussion mixes background and results. Improve by:First summarizing key findings, Then interpreting mechanisms with literature, Lastly discussing practical implications (energy, yield efficiency)

Author Response

RESPONSE to the Reviewer 3.

Dear Reviewer,

Kind regards,

The authors.

This manuscript investigates the effects of different full-spectrum LED lights (3000K, 4000K, 5000K) versus HPS on radish morphophysiology and leaf reflectance. The topic is timely and relevant to controlled-environment agriculture and lighting optimization. The study is well-structured and includes detailed spectral and physiological analysis. However, revisions are needed to improve scientific clarity, data interpretation, and organization.

The introduction is lengthy and mixes general CEA background with study-specific gaps. Clearly state the knowledge gap and novelty in last paragraph of introduction.
Add a sentence clearly stating why studying sun-like LEDs simulating midday solar spectrumis novel and how it advances radish lighting optimization.

Response 1: We greatly appreciate the reviewer's valuable suggestion. We have tried to rewrite the introduction in accordance with the comments of all the reviewers. In order to emphasize the novelty and originality of the research, as well as the knowledge gaps, we have added the following statements:

Lines 71-76: However, the role and mechanisms of the effects of the full spectrum of PAR on agricultural crops have not yet been fully understood. This knowledge would significantly expand the possibilities for managing plant productivity and quality. In this regard, it is important to develop LED modules with a full spectrum of radiation and select optimal spectra for growing various types of crops, in particular radish, taking into account the interaction between the genotype and the environment.

Lines 106-115: However, the relationship between light and the physiological state of plants, their productivity, and the spectral characteristics of diffuse leaf reflectance remains poorly understood in many crops, particularly radish. The purpose of this study is: 1) to study the effect of spectral radiation characteristics of HPS lamps and three full-spectrum simulating sunlight LED lamps with different ratios of physiologically active wavelengths in the PAR range on plants development and yield of two radish cultivars; 2) to evaluate the relationship between diffuse reflection indices of leaves and morphophysiological parameters of radish plants, in order to identify optical criteria for improving plant growing technologies and selecting economically valuable genotypes.

PPFD is controlled (good), but light intensity uniformity measurements across canopy not reported. Indicate whether light maps or measurements at multiple canopy points were taken. Provide PPFD uniformity details (e.g., min-max range, coefficient of variation).

Response 2: Thank you for your suggestion. We have made significant additions to Section 2.1. In addition, we have added Figures S1 and S2 for greater clarity.

Clarify whether analyses used n = 16 per treatment or n = 8 averaged over repetitions.

Response 3: Thank you for your suggestion. We tried to clarify:

Lines 171-174: The experiment was repeated twice. In each experiment, the number of plants evaluated was 8; since similar average values of the studied parameters were obtained in both experiments, statistical processing was performed for a combined sample of 16 radish plants.

Many figures are crowded, difficult to read, and not self-explanatory. Increase font size, Use consistent labeling, Include significance letters more clearly, Define abbreviations in captions (Ei, AI, LA, etc.)

Response 4: We greatly appreciate the reviewer's valuable suggestion. We performed additional statistical processing to improve the presentation of the results:

Lines 297-310: Statistical evaluation of the obtained data was performed by calculating the main descriptive characteristics: mean, and standard error of the mean (SEM). Duncan's multiple-range test was used to identify differences between mean values for each characteristic. A p value of < 0.05 (5% error probability) was considered an acceptable limit of statistical significance. Data analysis was performed using Microsoft Office Excel 2019 and Statistica v. 13.3 (StatSoft Inc., Tulsa, OK, USA). A two-way analysis of variance (ANOVA) of morphological traits was conducted to identify significant relationships between genotype, environmental conditions (light regime), and interfactorial interactions.

The manuscript suggests: Higher anthocyanins and PRI = photoprotection due to blue-rich light However: No pigment or chlorophyll lab validation was performed to confirm remote sensing indices.

Response 5: Thank you for your suggestion. You are right, we did not perform biochemical analyses in these experiments. However, we, as well as a number of other researchers, have repeatedly conducted such studies on a number of different crops, and high reliable correlations have been obtained between the reflection indices and the de facto content of pigments and other compounds:

Lines 94-102:

The authors who developed the formulas for normalized reflectance indices tested them on various plant species and compared with the results of biochemical assessment of chlorophyll - ChlRI [18], carotenoids - SIPI [19], anthocyanins - ARI [21,22] amount in leaves. Also, the relationship between the photochemical reflectance index – PRI and the epoxidation of pigments of the xanthophyll cycle, the efficiency of light use and the efficiency of photosynthesis were determined [19]. We have previously used these indices both in field studies and in the CEA for phenotyping and evaluating the dynamics of changes in biomass, pigment composition, and yield prediction of wheat and lettuce plants that are deficient in mineral nutrients and water [14-16,23].

We have provided relevant references in the text of the manuscript. In further work aimed at creating an optimal technology for growing radishes in CEA, we also plan to perform a biochemical analysis of the leaves.

The discussion mixes background and results. Improve by: First summarizing key findings, then interpreting mechanisms with literature, Lastly discussing practical implications (energy, yield efficiency)

Response 6: Thank you for your suggestion. We have tried to structure the text of the manuscript according to your requirements. We did not separate the "Discussion" into a separate section, as, in our opinion, this would make it very difficult to perceive the results. However, we tried to give a discussion after each significant result.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, thank you for the great improvements. I have only the following last comments:

Table 2, first coulmn of number: uniform the number of decimals.

Usually the american puntaction for the decimal is adopted, so decimal point should be used instead of the comma (European style) in an international scientific article. Check through the article and uniform because both the systems are used depending on the table.

Conclusion section still needs improvements. Please, retrieve and direclty report the final overall conslusions and perspectives of your results. in the current forms main results are reported, not main conclusions.

Some expression like (lines 661-663) "Although the differences were not significant, a trend of higher yields was observed in both replications of the experiment." are not appropriated for a concluson but for a Result section. And reporting all the significant numerical differences is appropriate for and abstract rather than conclusion.

In addition, the final lines of future perspectives can be made more readable and formal. For example, line 670 "They will also focus.." to who it refers to? it is not clear.

Author Response

Dear Reviewer,

We sincerely appreciate your valuable suggestion.

We would like to express our deepest gratitude to all the Reviewers who took the time to provide us with valuable feedback on our manuscript. The comments and suggestions have been extremely helpful in identifying areas for improvement and we are grateful for the support.

We hope that our findings will spark interest among the scientific community and contribute to the development of a technology for growing root vegetables, especially radishes, in controlled environments.

Kind regards,

Authors

Table 2, first coulmn of number: uniform the number of decimals.

Response 1: We greatly appreciate the reviewer's valuable suggestion. You are absolutely right, we made a technical error when designing Table 2. We have adjusted it:

Table 2. The proportion of commercial root crops and the yield of root crops of two radish cultivars under different lighting treatments.

Lighting treatment		Commercial root crops, %				Yield, kg m^-2
		cv. Pf		cv. P			cv. Pf		cv. P
HPS	95.0 ± 5.0		85.0 ± 5.0		3.7 ± 0.1			2.4 ± 0.2
LED 1	85.0 ± 5.0		75.0 ± 5.0		3.1 ± 0.2			1.9 ± 0.2
LED 2	95.0 ± 1.5		80.0 ± 5.0		3.4 ± 0.1			2.3 ± 0.1
LED 3	95.0 ± 0.0		85.0 ± 5.0		3.6 ± 0.1			2.5 ± 0.1

Note: Data are presented as mean ± standard error of mean (M ± SEM), n=2.

Cv. Pf – cultivar Peterburgskiy fioletovyy, cv. P – cultivar Perno.

Conclusion section still needs improvements. Please, retrieve and direclty report the final overall conslusions and perspectives of your results. in the current forms main results are reported, not main conclusions.

In addition, the final lines of future perspectives can be made more readable and formal. For example, line 670 "They will also focus.." to who it refers to? it is not clear.

Response 2: Thank you very much for your suggestion.

The conducted studies have shown that sunlike full-spectrum LED lamps are promising for growing radish under CEA conditions. It was revealed that the part of morphophysiological characteristics of both radish cultivars, as well as the reflectivity and diffuse reflectance spectra of the leaves, varied significantly depending on the lighting treatment. It was also found that a higher content of blue radiation in the LED spectrum had a stimulating effect on the development of radish root crops, contributing to an increase in the yield. The highest yield of root crops was obtained under LED 3 treatment with the highest content of blue light in the PAR spectrum. The research suggests that radish plants treated with LED 3 lamp had lower values of radiation reflected from the leaves compared to plants treated with other full-spectrum LED lamps. They also had the highest values of indices that characterize chlorophyll content. Subsequent research will be aimed at creating light environment that support the development of optimal photosynthetic systems of radish plants, which would increase radiation use efficiency. It is also planned to focus on improving methods for noninvasive monitoring of plant physiological status based on recording their optical characteristics.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully evaluated the authors’ responses . The authors have satisfactorily addressed the concerns raised and have significantly improved the quality, clarity, and scientific soundness of the manuscript. In its current form, the manuscript meets the standards of the journal. Therefore, I recommend acceptance of the manuscript.

Author Response

Dear Reviewer,

We sincerely appreciate your valuable suggestion. We would like to express our deepest gratitude to all the Reviewers who took the time to provide us with valuable feedback on our manuscript. Their comments have been extremely helpful in identifying areas for improvement and we are grateful for their assistance.

Kind regards,

Authors