Review Reports - Photosystem II Responses at the Whole-Potato-Leaf Level After Colorado Potato Beetle Feeding

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled'ROS Generation and Photoprotective Mechanism of Potato 2
Plants After Colorado Potato Beetle Feeding' is well structured. I appreciate the valuable work of authors. Kindly check the PDF and revise the minor corrections accordingly. Best wishes

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Grammatical mistakes were found. Kindly revise the whole manuscript by native english speaker

Author Response

Dear Reviewer,

We would like to thank you for dedicating the time and the effort to review our work and for the valuable comments and suggestions that we adopted in our revised manuscript.

Responses to your comments are on the attached pdf file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors

I read with great interest the manuscript by Ilektra Sperdouli et al., "ROS Generation and Photoprotective Mechanism of Potato Plants After Colorado Potato Beetle Feeding." The topic of the study is highly relevant and poorly understood. The authors attempted to answer the question of how the portion of the potato leaf not consumed by the Colorado potato beetle responds to the damaging effects of the leaf-eating insect at the level of photosystem 2. To this end, the authors conducted well-designed experiments. They showed that feeding on a small portion of the leaf by the Colorado potato beetle resulted in a decrease in the maximum photosynthetic efficiency (Fv/Fm) in the undamaged portion. This may be the result of photoinhibition caused by a decrease in the efficiency of the oxygen-evolving complex (OEC). However, the number of open reaction centers (qp) of PS2 did not change, but their functional efficiency decreased. A similar situation is possible when the non-photochemical quenching (NPQ) photoprotective mechanism is activated, which is accompanied by a decrease in singlet oxygen formation in the leaf portion undamaged by the Colorado potato beetle. NPQ simultaneously caused a slight increase in hydrogen peroxide production, which, according to the authors, acted as a signaling molecule. Based on this, it was concluded that in response to damage to a portion of the potato leaf, a signaling pathway involving 1O2 and H2O2 is triggered, acting as a defense mechanism against the leaf-eating insect.

I have no doubts about the presented experimental data and their interpretation, but the authors' hypothesis seems purely speculative. It is unclear how the protective mechanism of the two ROS manifests itself. The hypothesis could be more substantiated if the authors characterized some components of the cellular antioxidant system and their response to the Colorado potato beetle.

I believe that minor revisions are required before the manuscript can be accepted for publication.

Kind regards

Author Response

Dear Authors

Dear Reviewer,

We would like to thank you for dedicating the time and the effort to review our work and for the valuable comments.

Response: We share this view but our goal was to evaluate the responses of PSII to Colorado potato beetle feeding. Thus, taking into account your comment we changed the title to “Photosystem II Responses at the Whole Potato Leaf Level After Colorado Potato Beetle Feeding”.

I believe that minor revisions are required before the manuscript can be accepted for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigated “ROS Generation and Photoprotective Mechanism of Potato Plants After Colorado Potato Beetle Feeding.” The study has some significance, but there are also several issues that need to be addressed.

Line 21: The Latin name of the Colorado potato beetle should be provided in the abstract when it is first mentioned.

In the Results section, the authors first emphasize in Section 2.1 that there is clear spatial heterogeneity between the left and right sides of the leaf, but then subsequently use the “neighboring area of the feeding spot” and the “rest of the leaf area” as the main units of comparison. How do the authors demonstrate that the original left–right heterogeneity does not seriously interfere with the subsequent regional comparisons? In addition, the so-called “spatiotemporal heterogeneity” actually includes only two time points, namely before feeding and 20 min after feeding. Strictly speaking, this cannot be considered a true spatiotemporal dynamic analysis.

In Figure 1, the feeding spot shows Fv/Fm, ΦPSII, and ΦNPQ values of 0, whereas ΦNO is 1. This essentially indicates that the area has become mechanically damaged tissue or an empty region. Why, then, do the authors still discuss these pixel values within the same interpretative framework as the energy partitioning parameters of intact tissues? Is this physiologically meaningful?

In the Discussion, the directly measured results are actually limited to several PSII-related chlorophyll fluorescence parameters and DCF-DA imaging results. However, the authors construct a complete mechanistic model involving OEC impairment, donor-side photoinhibition, NPQ, long-distance H2O2 transport, JA vascular signaling, and interspecific differences among insects. There is a clear gap between the evidence and the conclusions. The authors should focus more closely on the experimental results and substantially narrow the scope of the discussion.

Although the authors mention an approximately 1:1 sex ratio, they do not clarify whether the individuals used in the bioassays were randomly selected, whether their sex was recorded, or whether differences in feeding behavior between males and females were considered.

Line 348: “Three to four leaves from xx”, was the sample size 3 leaves or 4 leaves?

The authors only measured ΦPSII and did not assess PSI-related parameters. Since PSII and PSI function in series in linear electron transport, changes in ΦPSII alone are insufficient to determine the specific site at which photoinhibition occurs, especially given that the authors further discuss H2O2 and defense signaling. Moreover, the Colorado potato beetle is a typical chewing herbivore that causes direct mechanical damage to leaf tissues. Tissue loss, local dehydration, cell structural disruption, and changes in leaf optical properties must therefore be taken into account. Under such circumstances, the observed changes in chlorophyll fluorescence parameters cannot be simply attributed to fine photoprotective regulation or signaling responses.

Author Response

Dear Reviewer,

We would like to thank you for dedicating the time and the effort to review our work and for the valuable comments. Below is our point-by-point response to your suggestions.

Line 21: The Latin name of the Colorado potato beetle should be provided in the abstract when it is first mentioned.

Response: The Latin name of Colorado potato beetle was provided.

Response: The observed spatial heterogeneity between the “left and right sides” of the leaf before feeding retained also and after feeding, as it is mentioned on lines 125-126, and it was independent of the heterogeneity at the “neighboring area of the feeding spot” and the “rest of the leaf area”, as it can be observed in Figure 1.

Response: We changed the phrase “spatiotemporal heterogeneity” to “spatial heterogeneity” but we believe it can be used even for two time points.

Response: We are discussing the values of the chlorophyll fluorescence parameters at the feeding spot to show that despite the fact that this area, as you mentioned, was damaged, the “neighboring area of the feeding spot” and the “rest of the leaf area” could respond to mechanical damage by enhancing ΦNPQ in order to decrease the non-regulated loss of energy (ΦNO) and avoid oxidative stress at the “neighboring area of the feeding spot” and the “rest of the leaf area”. This response can be considered an example of an efficient photoprotection mechanism.

Response: The impairment of the OEC is a direct measurement (Fv/Fo) and explains the reason of photoinhibition (Fv/Fm, direct measurement) that is documented to be donor side and not acceptor side photoinhibition (lines 228-234, 248-252). The increased photoprotective mechanism of NPQ (direct measurement) was sufficient not only to keep the same fraction of open reaction centres (qp, measured) but also to reduce ¹O₂ generation collaborating that the observed photoinhibition was not an acceptor side photoinhibition. Thus, no gap between the evidence and the conclusions exists. The only speculation in our manuscript is the discussion on lines 317-319 about the JA and hydrogen peroxide similar operating mode based on literature.

Response: The individuals used in the measurements were randomly selected (added on line 342).

Line 348: “Three to four leaves from xx”, was the sample size 3 leaves or 4 leaves?

Response: Four leaves were measured before feeding but three leaves after feeding because the insect on the fourth leaf did not began feeding during the period of the 20 min.

Response: We share these views but our goal was to evaluate the responses of PSII to Colorado potato feeding. Thus, taking into account your comments we change the title to “Photosystem II Responses at the Whole Potato Leaf Level After Colorado Potato Beetle Feeding”.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The author has answered my questions, and I have no further questions.