Skeleton Photoperiod Enhances Photosynthetic Yield in Celery via Circadian-Regulated Metabolic Coordination

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please see the attached PDF file with my comments embedded throughout.

Comments for author File: Comments.pdf

Author Response

My manuscript has improved.  I have 17 minor comments:   

1. I believe the most appropriate term is 'skeleton.' Please check and revise its usage throughout the text.

Thank you for your constructive comments. In response to your comments:

Skeleton Photoperiod Enhances Photosynthetic Yield in Celery via Circadian-Regulated Metabolic Coordination

2. Please revise the citation format according journal instructions

Thank you for pointing out the deficiencies. We have made multiple corrections and highlighted them in the manuscript.

 

3. While the experimental approach of the study is well conceived, I do not understand the rationale for modeling the responses. There does not appear to be a clear justification for applying a computational model.

Thank you for your constructive comments. In the introduction, we did not elaborate on the principles of model construction; instead, we integrated the construction principles into the discussion to describe and provide a schematic diagram of the gene regulatory network. Our model was developed based on this gene regulatory network diagram. The revised introduction section presents the corresponding rationale as follows:

Mathematical modeling is a tool that integrates mathematics and physiology to enhance our understanding of living systems. It is particularly valuable for elucidating the regulatory relationships among genes within the circadian clock, the output of regulatory signals from this biological clock, and the influence of environmental factors on the circadian oscillator [26–29,21]. Additionally, mathematical modeling provides a clear visualization of gene regulatory relationships and the effects of environmental changes on the biological clock. Common mathematical modeling approaches include Boolean models [30] and differential equation-based methods [28,29], both of which aim to capture the interrelated positive-negative feedback loops in different ways [31,32]. Boolean models simplify genes into on/off states and have been successfully employed to model circadian rhythms. Similarly, differential equation-based models have successfully reproduced the response of the Arabidopsis circadian clock to different light conditions [33].

 

4. I suggest explicitly providing the hypothesis of your study.

Thank you for constructive comments, we agree with you. In response to your comments:

In this study, we investigated the regulatory mechanism of Skeleton Photoperiod on photosynthetic yield using celery as the experimental material. By modeling differential equations, we simulated the endogenous circadian oscillations induced by changes in photoperiod and their cascading effects on photosynthesis. These effects included: (1) rhythmic expression of key photosynthetic genes (e.g., Lhcb1, psbA); (2) cyclic fluctuations in photosynthetic parameters (such as net photosynthesis rate and transpiration rate); and (3) variations in photosynthetic yield. Through a combination of model predictions and experimental validation, this study not only achieved accurate predictions of celery's photosynthetic yield under different photoperiodic conditions but also provided theoretical support for the optimal regulation of light environments in controlled cultivation systems.

5. I don't think it's appropriate to announce the results so early.

Thank you for your advice; we agree with your suggestions. We have removed that section and reorganized it as a conclusion in the conclusions section.

6. This section lacks basic information about the growing conditions necessary for the reproducibility of the study. For example, details such as temperature and humidity in the greenhouse, the type of substrate and pots used for plant cultivation, the fertilization regime, and the cultivation period should be provided.

Thank you for pointing out the inaccuracies, which we have detailed in Section 2.1. The modifications are as follows:

All 40 experimental celery seedlings ('Ningqin 1') were cultivated in a climatic greenhouse at Nanjing Agricultural University (longitude 188.84°E, latitude 32.04°N). All seedlings were planted in biodegradable pots measuring 10 cm x 10 cm x 10 cm, filled with a mixture of coir, vermiculite, and perlite (5:3:2). The temperature in the climate chamber was maintained at 22 ℃, with relative humidity ranging from 70% to 80%. CO₂ concentration was set at 400 µmol/mol, and the light source consisted of an LED cool light lamp providing a light intensity of 187.5 µmol/m²/s. The distance between the LED panel and the plants was maintained at 20 to 30 cm, and the plants were spaced 15 cm apart. The position of the seedlings was adjusted daily to ensure they received adequate light. Adjust the position of the seedlings daily to ensure they receive adequate light, and water them every three days to maintain sufficient moisture in the substrate.

7. How much is sufficient?

Thank you for your question. The light intensity in the climate chamber is set to 180 µmol/m²/s.

8. For this determinations, some details need to be provided: Chamber temperature, CO2 concentration, photosynthetic photon flux density, vapour presure deficit, and flow rate.

Thank you for your constructive comments. We have elaborated on them in Section 2.1.

9. As I understand it, and based on your results, the modeled responses closely resemble the experimental data. Therefore, it is unclear how the model contributes to a deeper understanding of the response mechanisms if it merely replicates what the experimental data already show. Moreover, your main objective was to investigate the responses of celery to skeleton photoperiods, not to develop a computational model. If your primary aim were the latter, the study’s approach would need to shift to emphasize the necessity of an accurate model for photosynthetic responses under abnormal photoperiods.

Thank you for your questions. In response to your inquiry, we have elaborated in the introduction. This elaboration is as in the response to the third question above.

10. Throughout the results section, there are some sentences that appear to belong in the discussion. In this section, you should focus solely on presenting the findings of your study, without providing explanations or interpretations at this stage.

Thank you for your valuable suggestions, which we have fully accepted. We have made all the necessary changes to the manuscript, including deleting or rewriting certain descriptions.

11. This belongs in the Discussion section, not in the Results section per se.

Thank you for highlighting the inaccuracies; we fully accept your suggestions. We have revised the section accordingly. The revisions are as follows:

Under 3 h light/3 h dark (3L:3D) cycle, Lhcb1 expression peaked at dusk and subsequently entered a period of low expression later in the day, characterized by multiple smaller peaks (see Fig.1A). Under 6 h light/6 h dark (6L:6D) cycle, the expression of Lhcb1 exhibited a peak at dusk, followed by a degradation during the night. There was a lower expression that rose more slowly during the second photoperiod and a smaller peak at dusk (see Fig. 1B). In Fig.1A and 1B, an interesting phenomenon is observed: two peaks were reached at dusk during the expression of Lhcb1. The second peak is lower than the first, and both peaks appear symmetrically. Surprisingly, the expression pattern of Lhcb1 under LL conditions was very similar to that observed under 12 h light/12 h dark (12L:12D) cycles (see Fig. 1C and D). From the results of the experiment, it is evident that the photosynthetic genes consistently exhibit a more robust circadian rhythm under different photoperiods. The dynamic results of Lhcb1 simulated by the model are consistent with those of the experiment, especially the phase, which indicates that the model has good qualitative adaptability.

12. Discussion

Thank you for pointing out the inaccuracies; we fully accept your suggestions. We have made changes to that section, which are included in our response to question 11.

13. Wher can I see this in the figure?

Thank you for your question. For the significance analysis, we have performed the statistical analysis in Materials and Methods 2.9, and only the error analysis is shown in the image.

14. I suggest avoiding affirmative headings in the Results section. They are more appropriate for the Discussion.

Thank you for your suggestions, which are always accepted. In response to your comments:

Photosynthetic parameters have different expression patterns in the Skeleton Photoperiod.

15. Please provide statistical evidence to support this assumption

Thank you for your query. We accept it. Our intention was to present a hypothesis for study below; however, the tone in the description of the question was overly positive, and it has been modified accordingly:

This finding suggests that changes in photoperiod can affect photosynthesis, especially at shorter photoperiods (3L:3D, 6L:6D), where photosynthesis may be greatly affected.

16. Could you provide statistical evidence to support this statement? I could not find a description of how the statistical analysis was performed to test the effect of the photoperiod. This should be clearly detailed in the Materials and Methods section, and corresponding p-values should be reported in the Results.

Thank you for your question. The amount of Pn accumulation is calculated by integrating the Pn curve. Subsequently, the accumulations under different photoperiods were analyzed comparatively. However, this calculation is not suitable for statistical analysis.

17. The conclusions should focus solely on the key findings of your study. In its current form, it reads more like a summary.

Thank you for your suggestions, which are always accepted. In response to your comments:

In this study, we obtained a set of optimal parameters related to the biological clock by constructing a differential equation model of celery's circadian clock and employing a cost function optimization method. We also conducted a systematic verification of the model's reliability. Based on the validated circadian clock model, we further developed a circadian clock pathway model to regulate photosynthesis, enabling the quantitative calculation of photosynthetic yield in celery. The results indicated that the 3L:3D photoperiod condition exhibited the best photosynthetic yield performance among all the tested photoperiods. To assess the generalizability of this finding, we extended the simulation to various photoperiods not included in the experiments (1L:1D, 1.5L:1.5D, 8L:16D, and 16L:8D), and the results consistently confirmed the significant advantage of the 3L:3D photoperiod. Statistical analyses of key photosynthetic parameters (Pn, Chl, and N) further supported these conclusions: under the 3L:3D photoperiod, the highest levels of Chl and N content were observed in the cells. Simultaneously, net Pn showed the strongest positive correlation with Chl and N, while exhibiting the strongest negative correlation with Ci. It is important to note that this study focused on the mechanism of photoperiodic influence on photosynthetic yield, without considering the interactions of other environmental factors such as light intensity, light quality, and temperature. The regulatory effects of these factors on photosynthesis warrant further exploration in subsequent studies.

This study offers a theoretical foundation and technical support for the photoperiod regulation of celery and other crops. The established modeling framework can be extended and applied to the investigation of circadian rhythms and photosynthesis in various crops. The findings of this research are highly significant for optimizing light management strategies in controlled environment agriculture.

18. All these statements are not conclusions of the study. Please revise

Thank you for your suggestions, which are always accepted. Same response as 17.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor and authors

The manuscript presents valuable research and addresses an interesting topic with potential relevance in the field. The authors have made a significant effort in conducting the experiments and calculating the simulations. However, there are important issues that need to be addressed.

The material and methods are not described precisely without details. So, this part should be expanded. The results are presented too extensively, there are elements of discussion in the results although there is a separate discussion chapter. The results need to be written more concisely, and the part related to the discussion needs to be integrated into the discussion. It is marked in the text.

The manuscript is quite difficult to follow, so data should flow from the materials and methods to the results and discussion. The structure of the manuscript is not clear.

The conclusion should be written in such a way that it contains the general conclusions of the work obtained from the conducted research.

All species names in Latin should be written in italics. The titles of the tables in the manuscript should be reformulated. So, they need to be short and concise and contain only the necessary information.

The citation in the text should be written in the form of numbers, making sure that they are linked properly with the references in the list of literature, because there are many omissions there.

All other remarks are in the manuscript.

Best regards,

Reviewer

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please improve the style of the English in the manuscript.

Author Response

My manuscript has improved.  I have 31 minor comments:   

1. Apium graveolens Italic.

Thank you for pointing out the inaccuracies, we agree. Several occurrences of proper names in the text have been italicized. These are shown below:

Apium graveolens L.  ,Arabidopsis thaliana  

2. Instead Roeber et al., 2022 it should be 11. Please, check the numbers of the references throughout the text.

Thank you for pointing out the problems, which we accept unanimously. The formatting of several references in the text has been adjusted, and all literature in the full text has been checked accordingly.

3. These two sentences are for the part RESULTS!

Thank you for your suggestion, which we agree with. The section has been revised to:

In this study, we investigated the regulatory mechanism of Skeleton Photoperiod on photosynthetic yield using celery as the experimental material. By modeling differential equations, we simulated the endogenous circadian oscillations induced by changes in photoperiod and their cascading effects on photosynthesis. These effects included: (1) rhythmic expression of key photosynthetic genes (e.g., Lhcb1, psbA); (2) cyclic fluctuations in photosynthetic parameters (such as net photosynthesis rate and transpiration rate); and (3) variations in photosynthetic yield. Through a combination of model predictions and experimental validation, this study not only achieved accurate predictions of celery's photosynthetic yield under different photoperiodic conditions but also provided theoretical support for the optimal regulation of light environments in controlled cultivation systems.

5. It should be written how many seedlings were per each treatment? Did you have repetitions?

Thank you for your question, we agree. The section has been provided with a new description:

All 40 experimental celery seedlings ('Ningqin 1') were cultivated in a climatic greenhouse at Nanjing Agricultural University (longitude 188.84°E, latitude 32.04°N). All seedlings were planted in biodegradable pots measuring 10 cm x 10 cm x 10 cm, filled with a mixture of coir, vermiculite, and perlite (5:3:2). The temperature in the climate chamber was maintained at 22 ℃, with relative humidity ranging from 70% to 80%. CO₂ concentration was set at 400 µmol/mol, and the light source consisted of an LED cool light lamp providing a light intensity of 180 µmol/m²/s. The distance between the LED panel and the plants was maintained at 20 to 30 cm, and the plants were spaced 15 cm apart. The position of the seedlings was adjusted daily to ensure they received adequate light. Adjust the position of the seedlings daily to ensure they receive adequate light, and water them every three days to maintain sufficient moisture in the substrate.

6. How old were the seedlings?

Thank you for your question, we agree. Question Response For:

Initially, all seedlings were cultured under a 12 h light 12 h dark (12L:12D) cycle for 5 days. They were then transferred to photoperiods of 3L:3D, 6L:6D, and 24L:0D for 6 days, respectively.

7. It is not clear. Did you have a treatment 12L:12D and did you leave the seedlings under this light regime together with other three light treatments? Or you sampled the leaves after 5 days for treatment 12L:12D?

Thank you for your question, we fully accept it. Question Response For:

Initially, all seedlings were cultured under a 12 h light 12 h dark (12L:12D) cycle for 5 days. They were then transferred to photoperiods of 3L:3D, 6L:6D, and 24L:0D for 6 days, respectively. The seedlings maintained under the 12L:12D condition were the control group and continued to be kept in this photoperiod.  Seedlings were sampled starting when they had 5 leaves (day 5 after transfer). Seedlings grown under 6L:6D, 12L:12D, and 24L:0D conditions had their leaves collected at 3-hour intervals following the light treatment. For seedlings grown under 3L:3D conditions, leaves were collected at 1.5-hour intervals after the light treatment, ensuring that the samples were not all collected at moments of alternating light and dark. ZT0 is the control time. All samples were collected in three replicates, then all samples were uniformly processed and immediately stored in an ultra-low temperature refrigerator at -80°C (Thermo Company, Waltham, MA, USA).

8. How do you collected the leaves after 10 days, if it is written that the plants were transferred to different light conditions for 6 days respectively?

Thank you for your question, we agree. Question Response For:

Initially, all seedlings were cultured under a 12 h light 12 h dark (12L:12D) cycle for 5 days. They were then transferred to photoperiods of 3L:3D, 6L:6D, and 24L:0D for 6 days, respectively. The seedlings maintained under the 12L:12D condition were the control group and continued to be kept in this photoperiod.  Seedlings were sampled starting when they had 5 leaves (day 5 after transfer). Seedlings grown under 6L:6D, 12L:12D, and 24L:0D conditions had their leaves collected at 3-hour intervals following the light treatment. For seedlings grown under 3L:3D conditions, leaves were collected at 1.5-hour intervals after the light treatment, ensuring that the samples were not all collected at moments of alternating light and dark. ZT0 is the control time. All samples were collected in three replicates, then all samples were uniformly processed and immediately stored in an ultra-low temperature refrigerator at -80°C (Thermo Company, Waltham, MA, USA).

9. Why did you sampled the leaves for this treatment differently? It should be explained.

Thank you for your question, we agree. Question Response For:

For seedlings grown under 3L:3D conditions, leaves were collected at 1.5-hour intervals after the light treatment, ensuring that the samples were not all collected at moments of alternating light and dark.

10. Leaves of how many plants? How many leaves per plant?

Thank you for your question, the question response is:

All 40 experimental celery seedlings ('Ningqin 1') were cultivated in a climatic greenhouse at Nanjing Agricultural University (longitude 188.84°E, latitude 32.04°N).

Seedlings were sampled starting when they had 5 leaves (day 5 after transfer).

11. X scale in the Figures is not clear 120-144 hours it is 24 h period of which day? the fifth day 120/24=5 And what period of the day did you start to measure?

Thank you for your question; the meaning of our X-axis has been redefined to use ZT0 as a reference point. In response to your comments:

12. 
    How did you calculate the relative expression level? It should be explained in the material and method.

Thank you for your question.  In response to your comments:

2.5. Real-time fluorescence quantitative (RT-qPCR) analysis

Total RNA was extracted from celery leaves using the Hi-Pure Total RNA Extraction Kit for Polysaccharide and Polyphenol Plants (TSP0202, Dynaeco). The integrity and purity of the RNA were assessed through 1.2% agarose gel electrophoresis. The concentration of the RNA samples was measured using a micro UV detector (Nanodrop ND-1000, Thermo Fisher Scientific, USA). Expression levels of photosynthesis-related genes associated with plant circadian rhythms were quantified using real-time quantitative PCR (RT-qPCR). Quantitative primers were designed using the online primer design tool Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Reverse transcription amplification was conducted with the DynaPro Reverse Transcription Kit SynScript® III RT SuperMix for qPCR. The cDNA products obtained from reverse transcription were diluted eightfold and used as templates for qPCR, which was performed with the DynaPro ArtiCanCEO SYBR qPCR Mix on the Bio-Rad CFX96 Real-Time PCR Platform. The amplification program consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s. The specificity of the amplified fragments was verified, and the melting curves were generated (95°C for 15 s; 65°C for 1 min; 95°C with a ramp rate of 0.1°C/s; 95°C for 15 s). Three biological replicates were performed, with BcGAPDH (BraC09g068080) serving as the internal reference gene for normalization. The differences in expression levels were calculated based on the Ct values using the  method [34], assuming an amplification efficiency of 100%.

13. The title of the table should be short and concise. This title is to long. There is no need to explain the Figures in the title. This text should be the part of the main text of theresults.

Thank you for your suggestion, we agree. In response to your comments:

Figure 1. Expression levels of Lhcb1 under different photoperiods. (A) Under 3L:3D conditions, the expression of Lhcb1 peaked in the morning and exhibited a multi-peak expression pattern in the afternoon. (B) Under 6L:6D conditions, Lhcb1 displayed a bimodal expression pattern in each cycle. (C) Under 12L:12D conditions, Lhcb1 expression peaked at noon and decreased to its lowest level at night. (D) Under continuous light (LL) conditions, the expression pattern of Lhcb1 resembled that observed under 12L:12D conditions.

14. The full mane of the abbreviation.

Thank you for your question. In response to your comments:

continuous light (LL)

continuous darkness (DD)

15. Same comments for fig. 2 as those for figure 1.

Thank you for your suggestion, we agree. In response to your comments:

Figure 2. Circadian patterns of Pn under different photoperiods. (A) Under the 3L:3D cycle, Pn exhibits a robust circadian rhythm. (B) Under the 6L:6D cycle, Pn displays a single peak within each cycle, with values dropping to their lowest point before the conclusion of the dark period. (C) Under the 12L:12D photoperiod, Pn peaks before noon and declines to a minimum during the late night. (D) Under LL conditions, Pn fluctuates gently, characterized by broader peaks and troughs.

16. Which test did you use to compare the content of Chlorophyll and nitrogen between the treatments? All about statistical methods used in the manuscript it should be written in the material and methods.

Thank you for your question, we fully accept it. Question Response For:

2.6. Measurement of Chl and N content in leaves

Celery seedlings were subjected to photoperiods of 3L:3D, 6L:6D, and 24L:0D for 5 days, during which the plants developed 5 leaves. Chl and N contents in the leaves were measured using a Konica Minolta Model 502 Portable Chlorophyll Analyzer (made in Japan). Additional measurements were taken on the 10th day of the seedlings under a 12L:12D cycle. Five biological replicates were performed for all measurements.

17. This part is for discussion, because you cite other paper.

Thank you for your suggestion, we agree. We have revised it.

18.This part for simulation should be explained in the material and method under 2.6. There should be written which simulations were done.

Thank you for your question, we fully accept it. Question Response For:

In the numerical simulation study, we not only reproduced the experimentally observed circadian expression patterns of photosynthetic genes and photosynthetic parameters, but also reproduced the photosynthetic yields under the four photoperiodic conditions used in the experiment. Additionally, we further simulated the photosynthetic yields under four photoperiods not included in the experiment (1L:1D, 1.5L:1.5D, 8L:16D, and 16L:8D), as well as the response of Skeleton Photoperiod genes to the circadian clock.

19. The same as for the figures 1 and 2.

Thank you for your suggestion, we agree. In response to your comments:

Figure 3. Accumulation of Pn under different photoperiodic conditions. (A) Under light-dark conditions, the accumulation of Pn gradually decreased with the prolongation of the photoperiod. Under continuous light (LL) conditions, Pn accumulation increased significantly, although it remained lower than the levels observed under 3L:3D conditions. (B) The Pn accumulation under long days, short days, and 1L:1D cycle treatments was comparable, and all were significantly higher than that observed under the 1.5L:1.5D cycle condition.

20. The citation of the other authors is for the Discussion.

Thank you for your question, we agree. We have revised it.

21. It is two long. Only title without this huge explanation.

Thank you for your suggestion, we agree. In response to your comments:

Figure 5. Patterns of relative changes in Pn and Ci. (A) Under the 3L:3D photoperiod, Pn exhibits a peak in the evening, while Ci reaches its peak at dawn, demonstrating a strong rhythmic pattern. (B) In the 6L:6D photoperiod, Pn peaks during the day, and Ci peaks after the dark period. (C) Under a 12L:12D natural photoperiod, Pn peaks during the day, while Ci minimizes during the day, with both parameters exhibiting relatively stable changes at night. (D) Under LL conditions, Pn peaks at 3 hours and subsequently remains low, whereas Ci shows an inverse pattern, with its minimum also occurring at 3 hours and then remaining elevated. All values are normalized to their respective maximum.

22. Not the surname of the author but number of the reference. And this part should be in the part of the discussion.

Thank you for pointing out the problems, which we accept unanimously. The formatting of several references in the text has been adjusted, and all literature in the full text has been checked accordingly. And, also make adjustments to that citation section.

23. Discussion.

Thank you for pointing out the inaccuracies; we fully accept your suggestions. Adjustments to this citation.

24. Nitrogen or N there is no need to put both.

Thank you for pointing out the problems, which we accept unanimously. In response to your comments:

Chl and N

25. This sentence should be deleted.

Thank you for your suggestion, we agree. It has been deleted

26. How did you calculate that relative level? It should be shown in the material and method.

Thank you for your question.  In response to your comments: Same response as 12

27. The title of the table should be short and concise. There is no need for all these comments. It should be in the text above.

Thank you for your suggestion, we agree. In response to your comments:

Figure 6. The interrelationships between Pn, Chl, and N. (A) Pn is significantly and strongly positively correlated with Chl and moderately positively correlated with N under a 3L:3D photoperiod. (B) Under a 6L:6D photoperiod, Pn exhibits a strong positive correlation with Chl and a moderate positive correlation with N. (C) Pn shows a moderate correlation with both Chl and N under a natural photoperiod of 12L:12D. (D) Under continuous light (LL) conditions, Pn is strongly positively correlated with both Chl and N. All values are normalized to their respective maximum.

28. Discussion.

Thank you for pointing out the inaccuracies; we fully accept your suggestions. Adjustments to this citation.

29. This should be at the end of the sentence.

Thank you for your suggestion, we agree. In response to your comments:

Skeleton photoperiod was originally used to entrain plant circadian clocks. An important hallmark of a functional circadian clock is its ability to be entrained by skeleton photoperiod[27], indicating that the impact of skeleton photoperiod on plant growth should not be overlooked. By precisely regulating photoperiodic parameters, it may be possible to achieve targeted improvements in crop agronomic traits, thereby significantly enhancing crop yields. As a common medicinal vegetable, if celery can optimize its lighting scheme based on Skeleton Photoperiod, it can not only increase its biomass and active ingredient content but also significantly enhance its economic value.

30.Two sentences begin same.

Thank you for your suggestion, we agree. In response to your comments: Same response as 12

31. These highlight sentences

Thank you for your suggestion, we agree. In response to your comments:

In this study, we obtained a set of optimal parameters related to the biological clock by constructing a differential equation model of celery's circadian clock and employing a cost function optimization method. We also conducted a systematic verification of the model's reliability. Based on the validated circadian clock model, we further developed a circadian clock pathway model to regulate photosynthesis, enabling the quantitative calculation of photosynthetic yield in celery. The results indicated that the 3L:3D photoperiod condition exhibited the best photosynthetic yield performance among all the tested photoperiods. To assess the generalizability of this finding, we extended the simulation to various photoperiods not included in the experiments (1L:1D, 1.5L:1.5D, 8L:16D, and 16L:8D), and the results consistently confirmed the significant advantage of the 3L:3D photoperiod. Statistical analyses of key photosynthetic parameters (Pn, Chl, and N) further supported these conclusions: under the 3L:3D photoperiod, the highest levels of Chl and N content were observed in the cells. Simultaneously, net Pn showed the strongest positive correlation with Chl and N, while exhibiting the strongest negative correlation with Ci. It is important to note that this study focused on the mechanism of photoperiodic influence on photosynthetic yield, without considering the interactions of other environmental factors such as light intensity, light quality, and temperature. The regulatory effects of these factors on photosynthesis warrant further exploration in subsequent studies.

This study offers a theoretical foundation and technical support for the photoperiod regulation of celery and other crops. The established modeling framework can be extended and applied to the investigation of circadian rhythms and photosynthesis in various crops. The findings of this research are highly significant for optimizing light management strategies in controlled environment agriculture.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors' efforts in addressing my comments. The article is now more coherent, and its presentation has significantly improved

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

you revised the manuscript according to our recommendations. I think it is more clear now, and it could be followed easily.

Best regards,

Reviewer