Cyclic Electron Flow Alleviates the Stress of Light Fluctuation on Soybean Photosynthesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript studies an interesting varying environmental condition that is the light discontinuity to which plants are naturally exposed and how these fluctuations affect photosynthesis. Their experimental design is very well thought and the measurements are adequate as they use known modern tools.

My concerns are about the interpretations derived from a non-objective analysis of the numerical differences or non-differences found, even though the statistical significance values clearly show small or not variations. This is explained in the following points.

1. Table 1. Lines 180-182. The text indicates differential effects of frequency fluctuations on chlorophyll and carotenoid determinations suggesting a sequence and decrease percentage. However, when statistical significance is observed the compared numbers are not necessarily different. A careful interpretation is recommended.

2. The design and panel composition of the figures are fine, however, they require more definition, since small numbers/letters look blurred and often are not easy to read.

3. Figure 3, lines 190-200. Photosynthetic parameters shown at panels a-f show differences, if any, very discrete. This is reflected by same letters indicating non-significant differences, which is a very common feature present in the different bars. In cases where more statistical significance is observed (see panels g-h), the differences among bars are magnified by the fact that the abscise axis is not departing from zero. Again. When the reader is referred to the text, description seems to depict differences that are significant, when they are not in the figures. It is important, when explaining percentage differences, to describe what is the 100 % value against to a given percentage value is calculated.

4. In the case of lines 200-204, which describe the effect that the frequency of fluctuation produces on the parameters ABS/CSm, TR0/CSm, ET0/CSm, and DI0/CSm obtained from the JIP curves in panel e, the authors describe “a decrease under long-term light fluctuation”, what it is fine, but an adjective as “slight” decrease really describes the magnitude of the observed effect  of the treatment on the J and I phases of the OJIP curve (see the the OJIP curves in panel e). I would not worry about the small differences found in photosynthetic parameters as a result of frequency of fluctuation, if measurements are well performed, and I think they are, this is the real biological response and its physiological meaning must be evaluated in these quantitative terms.

5. Figure 5, lines 213-236. The same arguments that I have explained in points 3 and 4, are applied to this figure. The description/interpretation of the results must be objective and sticked to the meaning of the statistical tests that were applied.

6. Figure 6. Discussion. The title of the figure starts improperly. The results shown indicate clear differences in the function of P700, however the numerical values are not very different among CK, IFL, 10FL and 20FL. The intention of this interpretation seems to justify/explain the results from figures 1-5. The results of Figure 6 are considered the most important as they support the title of the manuscript. I would say that an objective view of the overall data indicates that the effects of the frequency of variation of light in photosynthetic function are rather modest, which is a main finding and it agrees with the demonstration that photosynthetic apparatus is very robust and resilient to many forms of stress. The mechanism behind this resistance in the case of the variation of light exposure could be cautiously phrased as a suggested explanation.

Comments on the Quality of English Language

Only minor English details were found

Author Response

Comment1:

Lines 180-182. The text indicates differential effects of frequency fluctuations on chlorophyll and carotenoid determinations suggesting a sequence and decrease percentage. However, when statistical significance is observed the compared numbers are not necessarily different. A careful interpretation is recommended.

Response1:

Thank you for your valuable feedback. We have addressed the sequence of effects and emphasized the percentage changes in chlorophyll (Chl) and carotenoids. Additionally, we recognize the importance of careful interpretation when assessing statistical significance. Regarding the revised part, we have marked it in red on line 185-187 of the original text.

 

Comment2:

The design and panel composition of the figures are fine, however, they require more definition, since small numbers/letters look blurred and often are not easy to read.

Response2:

Thank you for your feedback. We appreciate your attention to detail. Based on your suggestion, we have improved the definition of small numbers and letters in the figures to enhance readability.

 

Comment3:

Figure 3, lines 190-200. Photosynthetic parameters shown at panels a-f show differences, if any, very discrete. This is reflected by same letters indicating non-significant differences, which is a very common feature present in the different bars. In cases where more statistical significance is observed (see panels g-h), the differences among bars are magnified by the fact that the abscise axis is not departing from zero. Again. When the reader is referred to the text, description seems to depict differences that are significant, when they are not in the figures. It is important, when explaining percentage differences, to describe what is the 100 % value against to a given percentage value is calculated.

Response3:

Thank you for your feedback. We acknowledge that the differences in photosynthetic parameters (panels a-f) are discrete, as indicated by the same letters denoting non-significant differences. To address this, we have clarified the significance levels in the revised text. Additionally, we recognize the importance of ensuring that the abscissa axis departs from zero in cases of greater statistical significance (as observed in panels g-h). Furthermore, we have explicitly described the 100% reference value against which percentage differences are calculated.

 

Comment4:

In the case of lines 200-204, which describe the effect that the frequency of fluctuation produces on the parameters ABS/CSm, TR0/CSm, ET0/CSm, and DI0/CSm obtained from the JIP curves in panel e, the authors describe “a decrease under long-term light fluctuation”, what it is fine, but an adjective as “slight” decrease really describes the magnitude of the observed effect  of the treatment on the J and I phases of the OJIP curve (see the the OJIP curves in panel e). I would not worry about the small differences found in photosynthetic parameters as a result of frequency of fluctuation, if measurements are well performed, and I think they are, this is the real biological response and its physiological meaning must be evaluated in these quantitative terms.

Response4:

Thank you for your insightful feedback. We appreciate your attention to detail. Based on your comments, we have made the following revisions: We acknowledge that the term “slight” may not fully describe the observed effect on the J and I phases of the OJIP curve(we have marked it in red on line 208-209 of the original text.). In the revised text, we have omitted the adjective and focused on the quantitative aspects of the biological response. We agree that the real biological response should be evaluated in quantitative terms. Our measurements were carefully performed, and we appreciate your emphasis on assessing physiological significance.

 

Comment5:

Figure 5, lines 213-236. The same arguments that I have explained in points 3 and 4, are applied to this figure. The description/interpretation of the results must be objective and sticked to the meaning of the statistical tests that were applied.

Response5:

Thank you for your meticulous review. We appreciate your insights, and based on your comments, we have made the following revisions: We have clarified the percentage changes relative to the control condition (CK) as the reference point (lines 220-223). We emphasized the substantial increases in Y(NPQ) under fluctuating light conditions without statistical significance (lines 224-225),to acknowledged the nuanced response of Y(NPQ) to varying light conditions. We concisely conveyed the lack of significant differences in Fv/Fm across treatments (lines 227-229) to provided a succinct summary of this parameter. We also summarized the impact of light fluctuation on Pm, Pm’, and Y(I) (lines 231-233) to highlighted the key findings related to photosynthetic performance.

 

Comment6:

Figure 6. Discussion. The title of the figure starts improperly. The results shown indicate clear differences in the function of P700, however the numerical values are not very different among CK, IFL, 10FL and 20FL. The intention of this interpretation seems to justify/explain the results from figures 1-5. The results of Figure 6 are considered the most important as they support the title of the manuscript. I would say that an objective view of the overall data indicates that the effects of the frequency of variation of light in photosynthetic function are rather modest, which is a main finding and it agrees with the demonstration that photosynthetic apparatus is very robust and resilient to many forms of stress. The mechanism behind this resistance in the case of the variation of light exposure could be cautiously phrased as a suggested explanation.

Response6:

Thank you for your insightful comments on Figure 6. We appreciate your attention to detail. Based on your feedback, we have revised the title of this section to better reflect the main findings. The updated title is ‘Modest Effects of Light Frequency Variation on Soybean Cyclic Electron Transfer.’(lines 272-273)

Regarding the numerical values, we agree that while clear differences in the function of P700 are evident, the actual numerical variations among CK, IFL, 10FL, and 20FL treatments are relatively modest. This aligns with our broader observation that the photosynthetic apparatus in soybeans exhibits robustness and resilience to various stressors. We appreciate your suggestion to cautiously phrase the mechanism behind this resistance as a suggested explanation.

Once again, thank you for your valuable input, and we believe these revisions enhance the clarity of our study.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors tested the effect of light fluctuation on soybean photosynthesis. The resolution of the figures appeared to low quality, resulting in fuzzy presentation as a pdf.  I also didn't find Figure 7 and it's legend to be very informative.  The authors need to explain what the red items indicate. Use of an arrow usually means flux through a pathway, but here they use red arrows which may mean less flow?  Also the use of a T-shape generally indicates that something is being blocked by that object, but here they may be implying something else. In addition some of the claims cannot be supported by the statistical analysis.  

Line 134- It is not clear what they mean by uniform growth.  Are they selecting plants that have the traits they expect?

Line 141 - The citation should probably be after the word test, and then replace the period with a comma.

Line 179 - All of the specific leaf weights are not statistically different.  Please remove all claims of them being greater from the manuscript. (Line 277, 352)

Line 182 - the 3.96% is not a statistically difference, please remove...

Line 290 - This reference doesn't match the text

Line 332, 348, 356, 372 refer to stimulating heat dissipation, but the authors provide no citations or support for this concept.  

Line 352 is not a complete sentence

Author Response

Comment1:

①The authors tested the effect of light fluctuation on soybean photosynthesis. The resolution of the figures appeared to low quality, resulting in fuzzy presentation as a pdf.

②I also didn't find Figure 7 and it's legend to be very informative.  The authors need to explain what the red items indicate. Use of an arrow usually means flux through a pathway, but here they use red arrows which may mean less flow?  Also the use of a T-shape generally indicates that something is being blocked by that object, but here they may be implying something else.

③In addition some of the claims cannot be supported by the statistical analysis.

Response1:

Thank you for your attention to detail. We appreciate your feedback.

①Figure Resolution:We have addressed the issue with the figure resolution, resulting in improved clarity. The revised figures now provide a higher-quality presentation.

②Example information for Figure 7: Arrowhead lines show the passageway, T-shaped lines show the passageway is blocked, and lines expand to show the passageway. The red image shows the area damaged, we have marked it in red on lines 378-380 of the original text.

③ Statistical Analysis: We recognize the need for additional support for certain claims. We will revisit our data and provide further evidence where necessary to strengthen the rigor of our study.

Your feedback is invaluable, and we remain committed to enhancing the clarity and robustness of our research.

Comment2:

Line 134- It is not clear what they mean by uniform growth.  Are they selecting plants that have the traits they expect?

Response2:

We carefully chose five soybean plants from the entire test treatment. These selected plants were morphologically uniform and representative, allowing us to conduct further detailed analyses.(Lines135-137)

 

Comment3:

Line 141 - The citation should probably be after the word test, and then replace the period with a comma.

Response3:

We have adjusted the citation to appear immediately after the word ‘test,’ replacing the period with a comma. Additionally, we included the phrase ‘as described in the literature’ for context. (Line144)

 

Comment4:

Line 179 - All of the specific leaf weights are not statistically different.  Please remove all claims of them being greater from the manuscript. (Line 277, 352)

Response4:

Thank you for your feedback. We have revised the manuscript accordingly, removing any claims of specific leaf weights being greater. The updated version now accurately reflects the statistical analysis. (Lines 185-187,358-359)

 

Comment5:

Line 182 - the 3.96% is not a statistically difference, please remove.

Response5:

Thank you for your feedback. We have revised the manuscript accordingly, removing the claim related to the -3.96% difference. The updated version now accurately reflects the statistical analysis.( Lines 188)

 

Comment6:

Line 290 - This reference doesn't match the text

Response6:

We appreciate the reviewer’s feedback regarding the reference. We will verify and correct any discrepancies to ensure accurate alignment with the text Special thanks to you for your good comments.

 

Comment7:

Line 332, 348, 356, 372 refer to stimulating heat dissipation, but the authors provide no citations or support for this concept. 

Response7:

Thank you for your feedback. We appreciate your attention to detail. We will address this concern by providing appropriate citations or support for the concept of stimulating heat dissipation in the revised manuscript.

 

Comment8:

Line 352 is not a complete sentence

Response8:

Thank you for your feedback. We appreciate your attention to detail. The revised sentence now accurately reflects the observation of a higher light-energy conversion rate in the photosynthetic system. ( Lines 359)