Light Stress Detection in Ficus elastica with Hyperspectral Indices

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The research in this article is very interesting, "Exposure of F. elasticity leaves to excess light (EL) (≥ 400 μ mol photons m-2s-1) results in an increase in reflection in the yellow green region (522-594 nm) and a decrease in reflection in the red region (666-682 nm) of the spectrum, company by a shift of the red edge point towards the longer wavelength. These changes were revealed using the previously proposed Light Stress Index (LSI=mean (R666:682)/mean (R522:594))...." Conducting stress assessment from the perspective of plant spectral measurement has significant research significance and value.

2. The abstract section is too complex, as it directly points out what problems we have addressed, what solutions we have proposed, and what results we have obtained. The expression in the article is too complex, and many unnecessary parts are presented here. Suggest summarizing again and rewriting the abstract section.

The format in Figure 2 is too arbitrary, for example, there is an error in the format between Figure 2 (b) and other figures; At the same time, please try to ensure that the starting points of the vertical axes of (a, b, c, d) 4 figures are consistent for readers to compare and analyze.

4. Figures 3 and 5 have similar issues as Figure 2, please modify them.

5. There are too many references cited in the discussion section. It is recommended to focus more on the scientific nature and progress of the experiments, and reduce the direct use of other people's work.

There are many formatting errors in the article, and we hope the author can make serious revisions.

7. The English expression throughout the article is poor and tense expression is unclear. We hope the author can further polish and revise the English throughout the article to ensure that it meets the requirements of the journal.

Comments on the Quality of English Language

The English expression throughout the article is poor and tense expression is unclear. We hope the author can further polish and revise the English throughout the article to ensure that it meets the requirements of the journal.

Author Response

Dear Reviewer!

Thank you so much for taking the time to review our manuscript! We appreciate your valuable feedback and constructive suggestions on our work. Your comments have made it much better. We retained revision marks on the revised manuscript in the ‘Manuscript_Revised_tracked changes.docx’ file. The authors hope, the current version of the manuscript will meet your expectations regarding clarity and quality of presentation.

 

1. The research in this article is very interesting, "Exposure of F. elasticity leaves to excess light (EL) (≥ 400 μ mol photons m-2s-1) results in an increase in reflection in the yellow green region (522-594 nm) and a decrease in reflection in the red region (666-682 nm) of the spectrum, company by a shift of the red edge point towards the longer wavelength. These changes were revealed using the previously proposed Light Stress Index (LSI=mean (R666:682)/mean (R522:594))...." Conducting stress assessment from the perspective of plant spectral measurement has significant research significance and value.

Response: Thank you so much for taking the time to review our manuscript!

 

2. The abstract section is too complex, as it directly points out what problems we have addressed, what solutions we have proposed, and what results we have obtained. The expression in the article is too complex, and many unnecessary parts are presented here. Suggest summarizing again and rewriting the abstract section.

Response: Thank you for your comment! The annotation has been revised. The changes can be tracked in a track-changed version.

 

3. The format in Figure 2 is too arbitrary, for example, there is an error in the format between Figure 2 (b) and other figures; At the same time, please try to ensure that the starting points of the vertical axes of (a, b, c, d) 4 figures are consistent for readers to compare and analyze.

Response: Thank you for your comment! We have made changes to the figure according to your comments. The changes can be tracked in a track-changed version.

 

4. Figures 3 and 5 have similar issues as Figure 2, please modify them.

Response: Thank you for your comment! We have made changes to the figures according to your comments. The changes can be tracked in a track-changed version.

 

5. There are too many references cited in the discussion section. It is recommended to focus more on the scientific nature and progress of the experiments, and reduce the direct use of other people's work.

Response: Thank you for your comment! We have tried to reduce the number of references and correct the text of the ‘Discussion’ section. The changes can be tracked in a track-changed version.

 

6. There are many formatting errors in the article, and we hope the author can make serious revisions.

Response: Thank you for your comment! We have endeavoured to correct formatting errors throughout the manuscript. The changes can be tracked in a track-changed version.

 

7. The English expression throughout the article is poor and tense expression is unclear. We hope the author can further polish and revise the English throughout the article to ensure that it meets the requirements of the journal.

Response: Thank you for your comment! We have revised the English language in the whole manuscript. We hope you will be satisfied with the quality of English in the current revision. The changes can be tracked in a track-changed version.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the use of hyperspectral indices to detect and quantify light stress in plants, focusing on Ficus elastica as a model species. The authors propose novel spectral indices for early detection of light stress and examine their effectiveness over 95 hours post-exposure to high-intensity light. This research has significant implications for advancing our understanding of plant physiology and improving agricultural & forestry management practices. However, there are several areas where the study could be strengthened to enhance its scientific rigor and broaden its applicability. I will elaborate on these points in detail:

 

1.        The limited number of replicates (three per treatment) raises concerns about the statistical power and reliability of the results. In plant physiology studies, particularly those involving spectral analysis, natural variability can be substantial. An analysis should be conducted to determine if the sample size is sufficient to detect meaningful differences between treatments.  How the potential limitations of the sample size may affect the interpretation of their results?

2.        The manuscript does not specify the developmental stage of the Ficus elastica plants used in the experiment. The plant response to light stress can vary depending on the plant's age and growth stage. Please clarify at what stage the experimental plants were and discuss how the findings might vary across different growth stages.

3.        While the use of LED lighting in controlled conditions is valuable for initial investigations, it's important to acknowledge that the spectral composition of LEDs differs from natural sunlight, particularly in the near-infrared region. This discrepancy could affect plant physiological responses and, consequently, the spectral signatures observed. Please address this limitation and, if possible, include validation under natural light conditions or using full-spectrum artificial lighting that more closely mimics sunlight.

4.        The preprocessing steps applied to hyperspectral data can significantly influence the results and interpretability. While the authors mentioned using a Savitzky-Golay filter for smoothing, more details would be beneficial. Please specify the window size and polynomial order used, and describe any bad band removal procedures applied.

5.        While the 95-hour post-exposure period provides valuable insights, it may not capture the full range of plant adaptation mechanisms. Consider discussing how seasonal variations in light intensity and duration might affect the applicability of your indices, and propose future experiments to examine longer-term responses.

6.        The manuscript presents valuable empirical data on spectral changes, but it could benefit from a deeper exploration of the underlying physiological mechanisms. How do the observed changes in reflectance relate to specific plant stress responses, such as changes in pigment composition, photosystem efficiency, or leaf structure?

7.        In natural environments, light stress often interacts with other environmental factors, such as temperature and water availability. How these interactions might affect the reliability and specificity of the proposed indices. Are there potential confounding effects that could limit the indices' applicability in field conditions where multiple stressors are present?

8.        The study effectively detects the presence of light stress, but further development of quantitative stress assessment would be valuable. Can the proposed indices be calibrated to provide quantitative measures of stress severity?

9.        The manuscript does not mention temperature control during the light exposure experiments. Given that high light intensities can lead to increased leaf temperatures, it's crucial to clarify whether and how temperature was regulated. If not, please add the content concerning the potential effects of heat stress on the results and how this might impact the interpretation of the light stress indices.

10.     Leaf age and position within the canopy can significantly affect spectral properties and stress responses. Please provide information on how leaves were selected for measurement and whether leaf age or position was standardized across treatments.

 

Author Response

Dear Reviewer!

Thank you so much for taking the time to review our manuscript! We appreciate your valuable feedback and constructive suggestions on our work. Your comments have made it much better. We retained revision marks on the revised manuscript in the ‘Manuscript_Revised_tracked changes.docx’ file. The authors hope, the current version of the manuscript will meet your expectations regarding clarity and quality of presentation.
Special thanks for your constructive comments and recommendations for future research!

 

This study investigates the use of hyperspectral indices to detect and quantify light stress in plants, focusing on Ficus elastica as a model species. The authors propose novel spectral indices for early detection of light stress and examine their effectiveness over 95 hours post-exposure to high-intensity light. This research has significant implications for advancing our understanding of plant physiology and improving agricultural & forestry management practices. However, there are several areas where the study could be strengthened to enhance its scientific rigor and broaden its applicability. I will elaborate on these points in detail:

Response: Thank you so much for taking the time to review our manuscript!

 

1. The limited number of replicates (three per treatment) raises concerns about the statistical power and reliability of the results. In plant physiology studies, particularly those involving spectral analysis, natural variability can be substantial. An analysis should be conducted to determine if the sample size is sufficient to detect meaningful differences between treatments. How the potential limitations of the sample size may affect the interpretation of their results?

Response: Thank you for your comment! Two-factor analysis of variance results have been added to the manuscript to address concerns about the statistical power and reliability of the results. It is shown that the influence of the factor ‘Sample Plant’ on the resulting trait is significantly lower than the influence of the factor ‘PPFD’. The results obtained are outlined in section ‘3.4.2 Results of analysis of variance’. The changes can be tracked in a track-changed version.

 

2. The manuscript does not specify the developmental stage of the Ficus elastica plants used in the experiment. The plant response to light stress can vary depending on the plant's age and growth stage. Please clarify at what stage the experimental plants were and discuss how the findings might vary across different growth stages.

Response: Thank you for your comment! The plants used in this experiment were obtained via tissue culture and grown in a greenhouse. All experimental plants were of the same age and had five developed leaves each on an unbranched stem. The plants were in the active growth stage. A detailed description of the experimental plants has been added to the Materials and Methods section on your recommendation. The changes can be tracked in a track-changed version.

Indeed, the growth stage of a plant (phenological state) is relevant to its response to light stress. We currently have no factual material on this issue and therefore do not discuss this in the manuscript.

 

3. While the use of LED lighting in controlled conditions is valuable for initial investigations, it's important to acknowledge that the spectral composition of LEDs differs from natural sunlight, particularly in the near-infrared region. This discrepancy could affect plant physiological responses and, consequently, the spectral signatures observed. Please address this limitation and, if possible, include validation under natural light conditions or using full-spectrum artificial lighting that more closely mimics sunlight.

Response: Thank you for your comment! A figure showing the spectrum of white LEDs between 450 and 950 nm has been added to section ‘2.2 Light Stress Induction’. In addition, we pointed out this problem in the section ‘Limitations and perspectives of the study’. The changes can be tracked in a track-changed version.

 

4. The preprocessing steps applied to hyperspectral data can significantly influence the results and interpretability. While the authors mentioned using a Savitzky-Golay filter for smoothing, more details would be beneficial. Please specify the window size and polynomial order used, and describe any bad band removal procedures applied.

Response: Thank you for your comment! Noise reduction is a critical preprocessing task in hyperspectral analysis. In this study the spectral profiles in the hyperspectral image were smoothed using a Savitzky-Golay filter with a 15 nm step size. The filter reduces the noise of the reflectance values by fitting a polynomial function and eliminates small differences between neighboring bands, which are most likely a result of measurement inaccuracy. The noiseFiltering method from the hsdar package for the R environment (Lehnert et al., 2019) was used for the calculations. No additional procedures to remove bad bands were applied.

Lehnert, L. W., Meyer, H., Obermeier, W. A., Silva, B., Regeling, B., & Bendix, J. (2019). Hyperspectral Data Analysis in R: The hsdar Package. Journal of Statistical Software, 89(12), 1–23. https://doi.org/10.18637/jss.v089.i12

 

5. While the 95-hour post-exposure period provides valuable insights, it may not capture the full range of plant adaptation mechanisms. Consider discussing how seasonal variations in light intensity and duration might affect the applicability of your indices, and propose future experiments to examine longer-term responses.

Response: Thank you for your suggestion! We will try to plan the design of such experiment.

 

6. The manuscript presents valuable empirical data on spectral changes, but it could benefit from a deeper exploration of the underlying physiological mechanisms. How do the observed changes in reflectance relate to specific plant stress responses, such as changes in pigment composition, photosystem efficiency, or leaf structure?

Response: Thank you for your suggestion! Yes, we are planning such experiments, in particular the use of electron microscopy to study changes in chloroplast dislocation under light stress.

 

7. In natural environments, light stress often interacts with other environmental factors, such as temperature and water availability. How these interactions might affect the reliability and specificity of the proposed indices. Are there potential confounding effects that could limit the indices' applicability in field conditions where multiple stressors are present?

Response: Thank you for your comment! Indeed, air temperature and soil moisture can modify the effect of light stress. This phenomenon is described in the study by Gitelson et al. (2024). In order to answer this question in relation to the proposed indices, we plan to conduct further multifactorial experiments.

Gitelson, A.A.; Zygielbaum, A.I.; Arkebauer, T.J.; Walter-Shea, E.A.; Solovchenko, A. Stress detection in vegetation based on remotely sensed light absorption coefficient. International Journal of Remote Sensing. 2024, 45:1, 259-277. https://doi.org/10.1080/01431161.2023.229583.

 

8. The study effectively detects the presence of light stress, but further development of quantitative stress assessment would be valuable. Can the proposed indices be calibrated to provide quantitative measures of stress severity?

Response: Thank you for your question! This study is essentially an exploratory experiment. The use of non-stressful PPDFs resulted in a rapid return to normal values for the VIs. The use of stressful PPDFs the effect persisted for a longer period of time. It is possible that the proposed VIs could be calibrated for the duration of effect retention, although this is not very convenient for practical use. In any case, further research is required.

 

9. The manuscript does not mention temperature control during the light exposure experiments. Given that high light intensities can lead to increased leaf temperatures, it's crucial to clarify whether and how temperature was regulated. If not, please add the content concerning the potential effects of heat stress on the results and how this might impact the interpretation of the light stress indices.

Response: Thank you for your remark! This is an important point that we missed when describing the methodology. The temperature increase under the action of different PPFD levels was only significant at 800 and 1200 μmol photons m^-2s^-1. (+ 4.9 and 6.7°C, respectively). The air was ventilated using a household fan to prevent overheating of the leaves. Section ‘2.2.1 Temperature control during light stress induction’ was added to the manuscript. The changes can be tracked in a track-changed version.

 

10. Leaf age and position within the canopy can significantly affect spectral properties and stress responses. Please provide information on how leaves were selected for measurement and whether leaf age or position was standardized across treatments.

Response: Thank you for your comment! The plants used for the experiment were juvenile ficus plants with 5 developed leaves on an unbranched stem. All leaves were formed under the same illumination. Therefore, the problem of leaf arrangement in the canopy, which is very important for adult plants, was absent in the presented experiment. The leaf age (also an important factor for the spectral characteristics of leaves) was strictly taken into account in the experiment. In all variants of the experiment, leaves of the same age - the third leaf from above - were used. This has been added to the ‘2.2 Induction of light stress’ section. The changes can be tracked in a track-changed version.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

General comments:

This study provides a comprehensive analysis of methods to detect plant stress using hyperspectral imaging, addressing key limitations such as species dependence, poor differentiation of stress types, and the challenge of early detection. The research focuses on Ficus elastica plants exposed to varying levels of photosynthetic photon flux density (PPFD) and examines changes in spectral reflectance as a result of excess light (EL). The study introduces two new Vegetation Indices (VIs), LSIRed and LSINorm, which, along with the previously proposed Light Stress Index (LSI), show a strong correlation with maximal quantum yields of photosystem II (Fv/Fm). The findings emphasize the persistence of stress effects under high PPFD levels and highlight the importance of considering light history in spectral monitoring of vegetation. Overall, it is a well-conducted study with significant contributions to plant stress detection, and I only have some minor comments, which are provided in my detailed feedback below.

 

Detailed comments:

Abstract: 

1. Line 11: how does this study address the first limitation? This limitation seems to be related to more sampling sites across different species and environmental conditions rather than development of spectral metrics? 

 

Introduction:

1. Line 40: abiotic stress is one of the main causes for yield loss?

2. Line 47-64: contents from this part to explain different mechanisms is meaningful but the writing is a little bit verbose.

3. You have too many paragraphs in the introduction part (normally 4-5 paragraphs). May combine related contents into one paragraph like Line 44-69.

4. Line 71: what does ‘high speed’ mean? High temporal frequency?

5. Line 78: the content of chlorophylls can be used as only abiotic stress, or does this also hold true for abiotic stress?

6. Line 83-86: there are two challenges. Distinguish biotic and abiotic stresses and distinguish different biotic stresses?

7. You may need to propose the research questions you want to address at the end of the introduction.

 

Methods:

1. Line 116: examples for light-loving species?

2. Table 1: why different experiments in different dates did not share the same PPFD level experimental set up?

 

Results and Discussion:

1. Please add units for the x and y axis for Figures 2 and 3.

2. Line 205: may use quantitative values to replace ‘significant changes’.

3. May also add the regression equation in Figure 5.

4. Figure 4: Some of the high correlation may be due to the similarity of the VI’s definition, which should be discussed.

5. So in this study, the abiotic stress was not directly measured but inferred from maximal quantum yields of photosystem II and photosynthetic photon flux density?

6. I suggested combining the result and discussion part as one part, since the discussion part is concise. For example, contents in Line 281-290 related to different VIs can be directly discussed after description contents in Figure 4. 

 

7. Line 320: what do you mean by “the physiological basis of the indices”, may need to provide some examples.

 

Comments on the Quality of English Language

Please see my previous comments. Thanks.

Author Response

Dear Reviewer!

Thank you so much for taking the time to review our manuscript! We appreciate your valuable feedback and constructive suggestions on our work. Your comments have made it much better. We retained revision marks on the revised manuscript in the ‘Manuscript_Revised_tracked changes.docx’ file. The authors hope, the current version of the manuscript will meet your expectations regarding clarity and quality of presentation.

 

This study provides a comprehensive analysis of methods to detect plant stress using hyperspectral imaging, addressing key limitations such as species dependence, poor differentiation of stress types, and the challenge of early detection. The research focuses on Ficus elastica plants exposed to varying levels of photosynthetic photon flux density (PPFD) and examines changes in spectral reflectance as a result of excess light (EL). The study introduces two new Vegetation Indices (VIs), LSIRed and LSINorm, which, along with the previously proposed Light Stress Index (LSI), show a strong correlation with maximal quantum yields of photosystem II (Fv/Fm). The findings emphasize the persistence of stress effects under high PPFD levels and highlight the importance of considering light history in spectral monitoring of vegetation. Overall, it is a well-conducted study with significant contributions to plant stress detection, and I only have some minor comments, which are provided in my detailed feedback below.

Response: Thank you so much for taking the time to review our manuscript!

 

Abstract: 

1. Line 11: how does this study address the first limitation? This limitation seems to be related to more sampling sites across different species and environmental conditions rather than development of spectral metrics? 

Response: Thank you for your comment! We have revised the text to include only species specifics. The changes can be tracked in a track-changed version. The development of species-specific universal VIs is theoretically possible. This is indicated by the studies of Féret et al. (2011) on the development of universal VIs and models for photosynthetic pigments.  In the future, we plan to test the VIs proposed in the manuscript on a larger set of plant species, primarily agricultural crops.

Jean-Baptiste Féret, Christophe François, Anatoly Gitelson, Gregory P. Asner, Karen M. Barry, Cinzia Panigada, Andrew D. Richardson, Stéphane Jacquemoud, Optimizing spectral indices and chemometric analysis of leaf chemical properties using radiative transfer modeling, Remote Sensing of Environment, Volume 115, Issue 10, 2011, Pages 2742-2750, ISSN 0034-4257, https://doi.org/10.1016/j.rse.2011.06.016.

 

Introduction:

1. Line 40: abiotic stress is one of the main causes for yield loss?

Response: Thank you for your comment! The suggestion has been corrected. The changes can be tracked in a track-changed version.

 

2. Line 47-64: contents from this part to explain different mechanisms is meaningful but the writing is a little bit verbose.

Response: Thank you for your comment! We have tried to shorten the text. The changes can be tracked in a track-changed version.

 

3. You have too many paragraphs in the introduction part (normally 4-5 paragraphs). May combine related contents into one paragraph like Line 44-69.

Response: Thank you for your comment! We have tried to reduce the number of paragraphs in the Introduction. The changes can be tracked in a track-changed version.

 

4. Line 71: what does ‘high speed’ mean? High temporal frequency?

Response: Thank you for your comment! Thank you for your comment! It was meant that remote spectral monitoring is more rapid than ground-based observations. The suggestion has been corrected. The changes can be tracked in a track-changed version.

 

5. Line 78: the content of chlorophylls can be used as only abiotic stress, or does this also hold true for abiotic stress?

Response: Thank you for your comment! Changes in chlorophyll content can be used to diagnose both abiotic and biotic stresses. The suggestion has been corrected. The changes can be tracked in a track-changed version.

 

6. Line 83-86: there are two challenges. Distinguish biotic and abiotic stresses and distinguish different biotic stresses?

Response: Thank you for your question! We are trying to identify specific responses to light stress as reflected in the spectral signature of plants.

 

7. You may need to propose the research questions you want to address at the end of the introduction.

Response: Thank you for your comment! We have made this part of the introduction more specific. The changes can be tracked in a track-changed version.

 

Methods:

1. Line 116: examples for light-loving species?

Response: Thank you for your comment! The paragraph has been corrected. The changes can be tracked in a track-changed version.

 

2. Table 1: why different experiments in different dates did not share the same PPFD level experimental set up?

Response: Thank you for your question! With a single hyperspectral camera, it is technically quite difficult to perform such an experiment correctly with 9 variants simultaneously. Besides, the experiment was exploratory in nature and it is more convenient to select PPFD stress levels in this way.

 

Results and Discussion:

1. Please add units for the x and y axis for Figures 2 and 3.

Response: Thank you for your comment! The figures have been corrected. In this case, the values of VI are unnamed quantities, so they do not have units on the y-axis. The changes can be tracked in a track-changed version.

 

2. Line 205: may use quantitative values to replace ‘significant changes’.

Response: Thank you for your comment! The text has been revised. The changes can be tracked in a track-changed version.

 

3. May also add the regression equation in Figure 5.

Response: Thank you for your comment! We have added the regression equation to the figure. The changes can be tracked in a track-changed version.

 

4. Figure 4: Some of the high correlation may be due to the similarity of the VI’s definition, which should be discussed.

Response: Thank you for your remark! The high strength of the correlation between the values of VIs can be explained both by the similarity of their formulas and their common physiological basis. The text has been revised. The changes can be tracked in a track-changed version.

 

5. So in this study, the abiotic stress was not directly measured but inferred from maximal quantum yields of photosystem II and photosynthetic photon flux density?

Response: Thanks for the question! Indeed it is.

 

6. I suggested combining the result and discussion part as one part, since the discussion part is concise. For example, contents in Line 281-290 related to different VIs can be directly discussed after description contents in Figure 4.

Response: Thank you for your comment! Your suggestion is a valid one. However, a review of articles in AgriEngineering journal shows that the separation of results and discussion is generally accepted. Therefore, we would like, if you do not object, to keep the same structure of the manuscript.

 

7. Line 320: what do you mean by “the physiological basis of the indices”, may need to provide some examples.

Response: Thank you for your question! The physiological basis of a VI is usually understood as a physiological process occurring in the plant, which is the root cause of the change in the value of this VI.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

No concerns from my side