Research on the Response Mechanism of the Photosynthetic System of Panax ginseng Leaves to High-Temperature Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Should ginseng really be italicized throughout?

Why is the symbol for protochlorophyll throughout ”Pch I” instead of ”Pchl”?

Page                    Line                      Comment

1                              41                          perhaps ”growth” instead of ”growing”.

2                              49                          ”high-” skip ”-”. See also line 2.

3                              101                       is ”light intensity” the correct term? I would prefer                                                                    ”illuminance”. See the following web addresses:

                                                                 https://fiveable.me/key-terms/principles-physics-iii-thermal-physics-waves/light-intensity

                                                                 According to https://en.wikipedia.org/wiki/Luminous_intensity

                                                                 lux is a unit for illuminance. For light intensity see

                                                                 https://en.wikipedia.org/wiki/Light_intensity

3                              113–114           ”then baked for 2 h until constant weight” Not clear: Was it                                              for 2 h or until constant weight?

3                              115                       ”calculated.Leaf” Insert space!

3                              115–116           ”(%) = (fresh leaf weight-dry leaf weight)/fresh leaf                                                                    weight×100%”. There must be one more parenthesis or                                              bracket: ”(%) = [(fresh leaf weight-dry leaf weight)/fresh                                                       leaf weight]×100%”

4                              134                       ”Protochlorophyll” should be ”protochlorophyll”.

4                              136                       Change ”fixed at” to ”adjusted to”!

4                              140–141           Change ”Take 0.1 g of fresh ginseng leaves that have                                                              undergone high-temperature treatment and place” to

                                                                 ” Fresh ginseng leaves (0.1 g) that had undergone high-                                                        temperature treatment were placed”.

4                              145                       a and b indicating forms of chlorophyll are often italicized: a,                                                                                                                                                                                           b.

4                              175                       ”um” should probably be ”mm” (as in the legend of Figure 6).

4                              177                       ”ultrathin sectioning machine”. Seems to be a very thin                                                       machine.        

4                              177–178           ”Leica” need not be repeated.

5                              182                       ”copper grid sections”; I prefer ”the copper grids with                                                           sections” since the copper grids were not sectioned.

6          Fig. 1 and Fig. 2.         Why are there lines between 48 and 0 hours

7                              249                       ”chlorophylase” should be ”chlorophyllase”.

                                                                 Also on line 267.                      

7                              253                       ”24.98% and 29.01%”. Are really 4 figures significant?                                                           Corresponding in some other places, e.g., lines 274, 281,                                                                                                                                                                                               287.

9                              296–297           ”As shown in Fig. 5a” … ”25.38%”. I could not see this.

10, 11.  Figures 6 and 7      The legends do not seem to fit these figures. They are no                                                    schematic diagrams.

 

Reference list

                                 Better leave a space before the last full stop (or leave out the last full stop) in the web addresses, so they can be clicked on directly.

                                 Reference 9 is perhaps not necessary, and ref. 14 less relevant in a heat context. Ref. 22 is not very relevant either. The Introduction is rather long and the number of references is rather large anyway.

Comments on the Quality of English Language

Should ginseng really be italicized throughout?

Why is the symbol for protochlorophyll throughout ”Pch I” instead of ”Pchl”?

Page                    Line                      Comment

1                              41                          perhaps ”growth” instead of ”growing”.

2                              49                          ”high-” skip ”-”. See also line 2.

3                              101                       is ”light intensity” the correct term? I would prefer                                                                    ”illuminance”. See the following web addresses:

                                                                 https://fiveable.me/key-terms/principles-physics-iii-thermal-physics-waves/light-intensity

                                                                 According to https://en.wikipedia.org/wiki/Luminous_intensity

                                                                 lux is a unit for illuminance. For light intensity see

                                                                 https://en.wikipedia.org/wiki/Light_intensity

3                              113–114           ”then baked for 2 h until constant weight” Not clear: Was it                                              for 2 h or until constant weight?

3                              115                       ”calculated.Leaf” Insert space!

3                              115–116           ”(%) = (fresh leaf weight-dry leaf weight)/fresh leaf                                                                    weight×100%”. There must be one more parenthesis or                                              bracket: ”(%) = [(fresh leaf weight-dry leaf weight)/fresh                                                       leaf weight]×100%”

4                              134                       ”Protochlorophyll” should be ”protochlorophyll”.

4                              136                       Change ”fixed at” to ”adjusted to”!

4                              140–141           Change ”Take 0.1 g of fresh ginseng leaves that have                                                              undergone high-temperature treatment and place” to

                                                                 ” Fresh ginseng leaves (0.1 g) that had undergone high-                                                        temperature treatment were placed”.

4                              145                       a and b indicating forms of chlorophyll are often italicized: a,                                                                                                                                                                                           b.

4                              175                       ”um” should probably be ”mm” (as in the legend of Figure 6).

4                              177                       ”ultrathin sectioning machine”. Seems to be a very thin                                                       machine.        

4                              177–178           ”Leica” need not be repeated.

5                              182                       ”copper grid sections”; I prefer ”the copper grids with                                                           sections” since the copper grids were not sectioned.

6          Fig. 1 and Fig. 2.         Why are there lines between 48 and 0 hours

7                              249                       ”chlorophylase” should be ”chlorophyllase”.

                                                                 Also on line 267.                      

7                              253                       ”24.98% and 29.01%”. Are really 4 figures significant?                                                           Corresponding in some other places, e.g., lines 274, 281,                                                                                                                                                                                               287.

9                              296–297           ”As shown in Fig. 5a” … ”25.38%”. I could not see this.

10, 11.  Figures 6 and 7      The legends do not seem to fit these figures. They are no                                                    schematic diagrams.

 

Reference list

                                 Better leave a space before the last full stop (or leave out the last full stop) in the web addresses, so they can be clicked on directly.

                                 Reference 9 is perhaps not necessary, and ref. 14 less relevant in a heat context. Ref. 22 is not very relevant either. The Introduction is rather long and the number of references is rather large anyway.

Author Response

Comments 1: Should ginseng really be italicized throughout?

Response 1: We fully agree with the reviewers' comments and express our gratitude.The Latin scientific name “Panax ginseng” in the title and manuscript line 40 is italicized according to standard formatting. All other references to ginseng in the text have been standardized and fully annotated to ensure uniformity.

Comments 2:  Why is the symbol for protochlorophyll throughout “Pch I” instead of ”Pchl”?

Response 2: We fully agree with the reviewers' comments and express our gratitude. We hereby apologize for the typographical error of miswriting protochlorophyll as “Pch I”. The manuscript has now been corrected to “Pchl”, and all relevant expressions in the text have been revised accordingly.

Comments 3: Line 41- perhaps“growth”instead of“growing”.

Response 3: We fully agree with the reviewers' comments and express our gratitude. We have already changed “growth” to “growing” in the manuscript.

Comments 4: Line 49 - “high-” skip ”-”. See also line 2.

Response 4: We fully agree with the reviewers' comments and express our gratitude. We modified the writing format of "high-" in the manuscript as required. After language polishing using journal-recommended editing software, the term “high-temperature” was rendered as “high-temperature”.

Comments 5: Line 101-is ”light intensity” the correct term? I would prefer ”illuminance”. See the following web addresses:https://fiveable.me/key-terms/principles-physics-iii-thermal-physics-waves/light-intensity According to https://en.wikipedia.org/wiki/Luminous_intensity lux is a unit for illuminance. For light intensity see https://en.wikipedia.org/wiki/Light_intensity

Response 5: We fully agree with the reviewers' comments and express our gratitude. We systematically organized the professional concepts related to illuminance and accordingly updated the manuscript by replacing “light intensity” with the more precise term “illuminance”.

Comments 6: Line 113-114 “then baked for 2 h until constant weight” Not clear: Was it for 2 h or until constant weight?

Pesponse 6: We fully agree with the reviewers' comments and express our gratitude. Due to language expression issues, I clarified whether the duration should be “Was it for 2 h or until constant weight”. Additionally, Reviewer 2 suggested changing “2 h” to “24 h”, and we made the corresponding revisions in the manuscript.

Comments 7: Line 115 “calculated.Leaf” Insert space!

Response 7: We fully agree with the reviewers' comments and express our gratitude. We have inserted a space between these two words in the manuscript.

Comments 8: Line 115-116 ”(%) = (fresh leaf weight-dry leaf weight)/fresh leaf weight×100%”. There must be one more parenthesis or bracket: ”(%) = [(fresh leaf weight-dry leaf weight)/fresh leaf weight]×100%”.

Response 8: We fully endorse the reviewers 'comments and extend our gratitude. In the manuscript, we revised the formula from “(%) = (fresh leaf weight-dry leaf weight) / fresh leaf weight × 100%” to “(%) = [(fresh leaf weight-dry leaf weight) / fresh leaf weight] × 100%”. Additionally, as noted by Reviewer 3 regarding the formula's phrasing, we have made modifications, the details of which are provided in section 3.2 of the manuscript.

Comments 9: Line 134 ”Protochlorophyll” should be ”protochlorophyll”.

Response 9: We fully endorse the reviewers 'comments and extend our gratitude. In the manuscript, we have revised “Protochlorophyll” to “protochlorophyll”.

Comments 10: Line 136  Change ”fixed at” to ”adjusted to”!

Response 10: We fully endorse the reviewers 'feedback and extend our gratitude. In the manuscript, we have revised “fixed at” to “adjusted to”.

Comments 11: Line 140-141 Change “Take 0.1 g of fresh ginseng leaves that have undergone high-temperature treatment and place” to ”Fresh ginseng leaves (0.1 g) that had undergone high-temperature treatment were placed”.

Response 11: We fully endorse the reviewers' comments and extend our gratitude. In the manuscript, we will revise the following “Take 0.1 g of fresh ginseng leaves that have undergone high-temperature treatment and place” change to “Fresh ginseng leaves (0.1 g) that had undergone high-temperature treatment were placed”。

Comments 12: Line 145 a and b indicating forms of chlorophyll are often italicized:a, b.

Response 12: We fully endorse the reviewers 'comments and extend our gratitude. In the manuscript, we have italicized “chlorophyll a, b”.

Comments 13: Line 175 ”um” should probably be ”mm” (as in the legend of Figure 6).

Response 13: We fully endorse the reviewers' comments and extend our gratitude. The image was designed to demonstrate the observation results at a 20 um scale under 2000× (×2.00 k means 2000×) magnification, as explicitly noted in the lower right corner of Figure 6.

Comments 14: Line 177 ”ultrathin sectioning machine”. Seems to be a very thin machine.

Response 14: We fully endorse the reviewers 'comments and extend our gratitude. To better visualize the structural changes in the tissue, we employed an ultrathin sectioning machine to slice ginseng leaves into 60-80 nm ultra-thin sections, which explains the necessity of this equipment. Additionally, we have corrected the terminology from “ultrathin sectioning machine” to “Ultra microtome” due to a phrasing issue.

Comments 15:  Line 177-178 “Leica” need not be repeated.

Response 15: We fully endorse the reviewers 'comments and extend our gratitude. The redundant "Leica" in the manuscript has been removed.

Comments 16: Line 182 “copper grid sections”; I prefer ”the copper grids with sections” since the copper grids were not sectioned.

Response 16: We fully endorse the reviewers 'comments and extend our gratitude. We have revised the manuscript to replace “copper grid sections” with “the copper grids with sections”.

Comments 17: Fig. 1 and Fig. 2. Why are there lines between 48 and 0 hours.

Response 17: We fully endorse the reviewers' comments and express our gratitude. The 48 and 0 h timeline is due to the inherent closed - loop nature of the radar chart. Given the radar chart's inherent limitations in fully presenting our findings, we have optimized the visual display for better clarity, as shown in Figures 1 and 2.

Comments 18: Line 249 and line 267. ”chlorophylase” should be ”chlorophyllase”.

Response 18: We fully endorse the reviewers 'comments and extend our gratitude. We have revised the term “chlorophylase” in lines 249 and 267 of the manuscript to “chlorophyllase”.

Comments 19: Line 253 “24.98% and 29.01%”. Are really 4 figures significant? Corresponding in some other places, e.g., lines 274, 281, 287.

Response 19: We fully endorse the reviewers 'comment and extend our gratitude. The statistical analysis methods and significance thresholds (e.g., P <0.05 for statistical significance) are clearly specified in the methods section of the manuscript. Data are presented as mean ± standard deviation (n≥3), with P <0.05 explicitly marked in the results section. All figures are uniformly labeled with significance markers (e.g., a/b/c lettering) to ensure statistical significance. Therefore, the percentages of 24.98% and 29.01% are statistically significant.

Comments 20: Line 296-297 ”As shown in Fig. 5 a” … ”25.38%”. I could not see this.

Response 20: We fully endorse the reviewers' comments and extend our gratitude. This sentence is the expression of the results of Figure 5. As shown in Fig. 5 a, high-temperature stress leads to a decrease in the content of chlorophyll a in ginseng leaves. Under high-temperature stress conditions of 35/24 ℃ and 40/30 ℃, the chlorophyll a content reached its minimum at 48 h, showing a relatively significant change as it decreased by 19.75% and 25.38%, respectively.

Comments 21: Figures 6 and 7 The legends do not seem to fit these figures. They are no schematic diagrams.

Response 21: We fully agree with the reviewer's comments and thank you. We have revised the legend as follows: Figure 6. The effect of high temperature stress on gaseous parameters of ginseng stomata. (a) Stomatal parameter of ginseng leaves at 25/16 ℃ (20 um, ×2.00k (2000×)); (b) Stomatal parameters of ginseng leaves at 40/28 ℃ high temperature stress for 48 h  (20 um, ×2.00k (2000×)).

Figure 7. The effect of high temperature stress on the ultrastructure of ginseng chloroplasts. a,b. Ultrastructural of chloroplasts in ginseng leaves at 25/16 ℃ (CK, 10 um); c,d. Ultrastructural of chloroplasts in ginseng leaves at 40/28 ℃ (high temperature stress for 48 h, 20 um). Note: The magnification of images a and c is ×1.0 k (1000×), and that of images b and d is ×4.0 k (4000×). Chl: chloroplast; GL: Grana; OG: Osmophilic granules.

Comments 22: Reference list: Better leave a space before the last full stop (or leave out the last full stop) in the web addresses, so they can be clicked on directly.

Response 22: We fully agree with the reviewer's comments and thank you. We have omitted the last period before the web address in the reference list to make it easier for you to click directly.

Comments 23: Reference 9 is perhaps not necessary, and ref. 14 less relevant in a heat context. Ref. 22 is not very relevant either. The Introduction is rather long and the number of references is rather large anyway. The Introduction is rather long and the number of references is rather large anyway. 

Response 23: We fully endorse the reviewers 'comments and extend our gratitude. We have removed References 9, 14, and 22, and revised both the introduction and the reference list. The specific modifications are detailed in the manuscript's preface and references section.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I think that your manuscript presents a valuable investigation into how elevated temperature affect ginseng leaf physiology and structure. I have provided my suggestions in the attached document, organized by sections, and I hope they will help you improve the manuscript.

 

 

 

 

Comments for author File: Comments.pdf

Author Response

Comments 1: Introduction:

Although the introduction provides background information on chlorophyll biosynthesis and the general sensitivity of ginseng to high-temperature stress, it does not clearly identify the specific knowledge gap or explain why this study is necessary. The novelty remains unclear: Is this the first comprehensive assessment for this species, the first integration of biochemical, physiological, and ultrastructural traits, or the first evaluation across the selected temperature regimes? The rationale for examining the chosen traits (chlorophyll biosynthesis enzymes, photosynthetic characteristics, stomatal morphology, and chloroplast ultrastructure) is not explicitly connected to previously established physiological or ecological concepts. In addition, the introduction does not provide a clear statement of the main research aim, nor does it specify hypotheses or testable predictions. Without explicit hypotheses, it becomes difficult for the reader to understand the interpretative framework used for the subsequent results.

Response 1: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. The introduction section of the manuscript has been revised, as detailed in the revised introduction.

Comments 2: Material and methods

Although plants were potted, provenance (source, genotype, nursery) should be provided; if wild material or protected access was used, permits should be declared.

Response 2: We fully endorse the reviewers' valuable suggestions and extend our gratitude. We have supplemented the manuscript with the sources of the experimental plant material, as follows: Three-year-old ginseng plants (Fushun County Ginseng Plantation Base, Baishan, China) were used in the experiment. 

Comments 3: Several descriptions are incomplete (e.g., number of biological replicates, how plants were selected, growth provenance), which prevents replication.

Response 3: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. The relevant sections have been revised as follows: Thirty ginseng plants were used in each high-temperature treatment group. Samples were randomly collected 12 h, 24 h, 36 h, and 48 h after high-temperature treatment and stored at -80 ℃ for subsequent use. Three independent biological replicates were performed at each time point, and each biological replicate underwent three technical replicate determinations.

Comments 4: It is unclear whether experimental units are individual plants or growth chambers; if all plants at a given temperature were housed in a single chamber, the study may suffer pseudoreplication. Details on how treatments were assigned and whether treatments were simultaneous are missing.

Response 4: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. The relevant section has been updated with the following clarification: Thirty ginseng plants were used in each high-temperature treatment group, and different growth chambers were used. Samples were randomly collected 12 h, 24 h, 36 h, and 48 h after high-temperature treatment. This design ensures experimental replication through independent growth chambers rather than sharing the same chamber, effectively preventing pseudo-replication issues.

Comments 5: Light is given in lux (Lx) instead of PPFD (µmol photons m⁻² s⁻¹), and temperature formatting should be standardized (e.g., 40/28 °C).

Response 5: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. The illumination intensity in the manuscript is expressed in Lx, and the temperature values have been standardized in accordance with the prescribed format.

Comments 6: Watering regime/pot soil moisture and vapour pressure deficit are not described, so it is impossible to separate direct heat effects from secondary drought effects.

Response 6: We fully endorse the reviewers' valuable suggestions and extend our sincere gratitude. During the experiment, the artificial climate incubator maintained humidity levels between 40% and 60%. This stable air humidity, closely correlated with soil moisture, effectively minimized fluctuations in soil evaporation and plant transpiration. At the same time, we added the following to the materials: During the experiment, soil moisture was monitored daily using a soil moisture detector (QS-SFY-I soil moisture rapid measuring instrument, Shanghai, China), with the moisture level maintained between 40 and 50% and water administered as needed.

Comments 7: Many instruments and assays are named, but the exact operating parameters (LI-6400 settings, CO₂ concentration, PAR, dark adaptation conditions, kit protocols, normalization) are absent.

Response 7: We fully appreciate the reviewers' valuable feedback and thank you. We have revised the relevant section, as detailed in the manuscript.

Comments 8: For enzyme activities and biochemical assays there is no mention of protein normalization, use of blanks/standards, calibration curves, or detection limits.

Response 8: We fully appreciate the reviewers' valuable feedback and thank you. We have revised the relevant section, as detailed in the manuscript. We have revised this section of the manuscript, with specific modifications detailed in the supplementary materials. As this study primarily focuses on the effects of high-temperature stress on the photosynthetic system of ginseng leaves, and emphasizes mechanisms related to photosynthesis, protein normalization data were excluded from the main text to maintain the integrity and focus of the manuscript. Given that the protein content data remains valuable for reference, it has been submitted as supplementary material for review.

Comments 9: Microscopy sampling & quantification insufficiently described. For SEM/TEM the sampling protocol, number of stomata/chloroplasts measured per leaf, and whether quantification was blinded must be stated.

Response 9: We fully endorse the reviewers' valuable suggestions and extend our sincere gratitude. Sampling protocol: The tissue on both sides of the central vein in ginseng leaves was cut into 1mm×3mm sections. Currently, the thickness of the thylakoid layer and the number of the granule stacks have not been measured in this study, so the quantitative analysis is not involved. We also agree with the research value of quantitative analysis, so the opinions of measuring the thickness of thylakoid layer and the number of particles are added as the limitation of the research in the discussion.

Comments 10: The statistical analysis only states that a “single factor and standard deviation” method was used. There is no description of model structure (factors, interactions), assumption checks, post-hoc testing, or handling of repeated measures (time points).

Response 10: We fully endorse the reviewers' valuable feedback and extend our gratitude. The revisions to section 2.9 are as follows: Data analysis was performed using SPSS 19.0 (IBM Corporation, Chicago, IL, USA). The results are expressed as the means of three independent measurements. Statistical evaluation of the experimental data was performed using a one-way ANOVA followed by Duncan’s multiple range test. A difference was considered statistically significant when P < 0.05. The results were graphically depicted using the Prism 8.0 (GraphPad Software, San Diego, USA) and Origin Pro 2021 (OriginLab Corporation, Northampton, MA, USA) software.

Comments 11:Results

Throughout the Results, the authors interpret physiological mechanisms, speculate about enzyme conformation, draw causal conclusions, and explain biological processes. This is inappropriate for the Results; such interpretations belong in the Discussion.

Response 11: We fully endorse the reviewers' valuable feedback and extend our gratitude. In the results section, we have incorporated these interpretations into the discussion section.

Comments 12: Nearly every subsection repeats that high temperature “significantly reduced,” “inhibited,” “weakened,” “damaged,” etc. Results should be more concise and focus on numerical outputs rather than repeating general conclusions.

Response 12: We fully endorse the reviewers' valuable feedback and extend our gratitude. The research findings section has been revised, with the updated content now emphasizing the description of research data.

Comments 13: The Results lack essential information about: Type of statistical test used (ANOVA? repeated measures? mixed model?); Correction for multiple comparisons; Effect sizes; Exact P values (instead of just P < 0.05 or P < 0.001); Whether assumptions were met; Whether interactions (temperature × time) were tested; Without this information, it is hard to judge the robustness of the results.

Response 13: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. In section 2.10 of the Materials and Methods, we have supplemented the description of the experimental data analysis methodology, and further expanded the graphical content in section 3.2, with the following specific additions: Data are presented as mean ± standard deviation (n ≥ 3). Different lowercase letters indicate significant differences at the P < 0.05 level. Furthermore, Reviewer 1 provided feedback on Figures 1 and 2. Recognizing the limitations of the original figures in presenting the research findings, we revised Figures 1 and 2 and re-presented the data using more appropriate graphical formats.

Comments 14: Techical issues: RWS is used instead of RWC in some sentences

Temperatures should be written with space before °C

Response 14: We fully agree with the reviewers' valuable suggestions and thank you. First, we sincerely apologize for the inconvenience. We have corrected the RWS in the manuscript to RWC and added a space before ℃ to standardize the temperature values.

Comments 15: Section by section comments: 

3.1. Only one temperature appears to cause significant reduction; statistical analysis not provided. Overinterpretation: “accelerated evaporation through stomata” - not measured.

Response 15: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. 3.1 The study demonstrates that high-temperature stress induces varying degrees of water content reduction in ginseng leaves. We revised Table 1 to present data as mean ± SD with statistical significance labels, providing a clearer visualization of relative water content (RWC) decline under different heat treatments. While RWC decreased in all high-temperature groups compared to the control, the 40°C treatment showed the most significant reduction. The manuscript's reference to “pore-mediated accelerated evaporation” as a potential mechanism has been removed. 

Comments 16:  3.2. Claims that the reduction of Pn is “mainly due to non-stomatal factors” require evidence such as chlorophyll fluorescence or mesophyll conductance modeling. Ci increase + Gs decrease does not automatically prove non-stomatal limitation.

Response 16: We fully endorse the reviewers' valuable feedback and extend our gratitude. We have revised the statement regarding the reduction of photosynthetically active radiation (Pn), which was previously attributed primarily to non-potassium factors, as follows: Ci increased as Gs decreased, and Pn was reduced, which is consistent with previous studies. This trend of change may be caused by the decrease in photosynthetic activity of mesophyll cells, driven by non-stomatal factors.

Comments 17: 3.3. Lack of clarity on whether photoinhibition is reversible. Increased Fo requires caution; could also be due to state transitions or antenna uncoupling.

Response 17: We fully endorse the reviewers 'valuable suggestions and extend our sincere gratitude. Your recommendation to relocate these conclusions to the discussion section has been duly implemented. We apologize for the previously inaccurate phrasing regarding' photoinhibition' and have made the necessary corrections. Furthermore, we have revised the explanation for the elevated Fo value as follows: High temperature leads to the decoupling of the PSII antenna pigment system and inactivation of the reaction center, thereby causing an increase in Fo values. 

Comments 18: 3.4. More numeric detail is needed (mean ± SD). Mechanistic statements such as“disrupt conformation of ALAD” or “metabolic homeostasis disrupted” are speculative. Need to indicate whether enzyme data are normalized to protein content.

Response 18: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. We have updated the figure captions for the manuscript's research data to read: Data presented as mean±standard deviation (n≥3). Regarding mechanistic descriptions such as “conformational disruption of ALAD” or “metabolic homeostasis disruption”, we have revised them to more accurately reflect the experimental findings. The enzyme activity data were standardized according to protein content and supplemented in the supplementary materials.

Commets 19: 3.5. Interpretation dominates over data. The narrative about “blockage of downstream metabolism” requires experimental evidence on enzyme activity beyond ALAD/PBGD.

Response 19: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. The primary objective of this study was to investigate the effects of high-temperature stress on the chlorophyll biosynthesis pathway. We selected ALAD and PBGD as the target enzymes, given their established role as rate-limiting enzymes in this pathway. Our experimental design was therefore specifically designed to examine the sensitivity of these two key chlorophyll enzymes to elevated temperatures. Research results show that ALAD activity decreases by 33.24% after 48 h of high-temperature stress at 40/28 ℃. The activity of PBGD was reduced by 29.01% after 48 h of high temperature stress at 40/28 ℃. At the same time, we measured the content of chlorophyll precursor substances. The synthesis of Mg-ProtoIX was reduced, which confirmed that high temperature inhibits the activity of ALAD and PBGD, seriously blocking the conversion of ProtoIX to Mg-ProtoIX, leading to a large accumulation of ProtoIX in the body. Due to the inhibited activity of ALAD and PBGD, the precursor of Pchl synthesis was reduced, ultimately causing a significant decrease in Pchl content in the later stage of high temperature stress. Ultimately, the observed reduction in chlorophyll content supports the conclusion that high temperatures inhibit chlorophyll synthesis by suppressing ALAD and PBGD. We concur with the reviewers' assessment, and this limitation will be addressed in the discussion section. Future studies will focus on measuring other enzymes involved in the chlorophyll biosynthesis pathway.

Comments 20: 3.6. Needs more precise statistical description.

Response 20: We fully endorse the reviewers' valuable feedback and extend our gratitude. The content has been supplemented, as detailed in section 3.6 of the manuscript.

Comments 21: 3.7. How many stomata measured per leaf? Was stomatal density assessed? Interpretation onosmotic potential, guard cell water loss belongs in Discussion.

Response 21: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. All leaf samples underwent measurement of six stomata each. While stomatal density remains unmeasured in this study (as our primary focus is evaluating high-temperature stress effects on ginseng leaf stomatal morphology), we have documented stomatal morphology images along with key parameters including length, width, and area. The stomatal density measurement will be incorporated into our future research plan. Furthermore, the analysis of osmotic potential and water loss in guard cells has been relocated to the discussion section.

Comments 22: 3.8. Descriptions should be more quantitative (e.g., thickness of thylakoid layers, number of granum stacks). The term “mitochondrial damage” is used, but mitochondria are not shown or analyzed quantitatively.

Response 22: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. As this study primarily investigates the effects of high-temperature stress on ginseng leaf chloroplast structure, we did not measure parameters such as thylakoid membrane thickness or basal body stacking quantity. These limitations have been addressed in the discussion section. The term' mitochondrial damage' was removed from the original text due to terminological inaccuracies.

Comments 23: 3.9. The direction and strength (r values) of correlations are not reported. Correlation heatmap interpretations are minimal; correlations do not imply causation. The heatmap figure is unclear; it is too small to be legible.

Response 23: We fully endorse the reviewers' valuable feedback and extend our gratitude. We have revised Figure 8, re-evaluated the correlation heatmaps, and now clearly indicate the direction and magnitude of correlations (r-values) in the plot labels.

Comments 24: Discussion

The authors frequently uses strong, mechanistic phrases such as: “irreversible damage”, “structural integrity destroyed”, “dual mechanisms jointly lead to...”, “cutting off the normal supply of metabolic substrates”, “hinders conversion process of chlorophyll precursors”... However, the study did not: conduct recovery experiments to show irreversibility, measure degradation pathways,quantify membrane protein damage, measure pigment-degrading enzymes or ROS. Thus, these claims should be softened or removed.

Response 24: We fully endorse the reviewers 'valuable feedback and extend our gratitude. Your suggestion to incorporate the conclusive findings from the research results section into the discussion has been implemented, with corresponding revisions made to the discussion section. The sections containing inappropriate conclusions have been removed, as detailed in the manuscript's discussion.

Comments 25: Large parts of the Discussion rely almost entirely on external literature (e.g., peony, cucumber, lily, blueberry) rather than integrating its specific findings. I Suggest starting with key results and then contextualizes them within the literature.

Response 25: We fully endorse the reviewers' valuable feedback and express our gratitude. We first discuss the key research findings, then elaborate on them in relation to the relevant literature. Specific revisions are detailed in the discussion section.

Comments 26:  The text claims that high temperatures “accelerate chlorophyll biosynthesis” (citing ref. 38), but also that ALAD/PBGD activities decrease and synthesis is blocked. These statements contradict each other unless explained carefully.

Response 26: We fully agree with the reviewer's valuable comments and thank you. We have rephrased the relevant content and revised the discussion section to avoid contradictions in wording.

Comments 27:  Several conceptual linkages are vague: How do decreased RWC and stomatal closure quantitatively relate to Pn decline? Are ALAD/PBGD decreases large enough to explain pigment loss? Do chloroplast ultrastructure changes precede or follow pigment decline? The Discussion needs a more coherent mechanism.

Response 27: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. In summary, the research results show that under high-temperature stress, the RWC of ginseng leaves decreases and the turgor pressure of guard cells drops, leading to stomatal closure, which limits the supply of CO₂ and reduces the Pn parameter. Meanwhile, RWC reduces the occurrence of cellular dehydration, which damages chloroplast ultrastructure. It inhibits chlorophyll biosynthetic enzymes ALAD and PBGD, disrupting the normal accumulation of chlorophyll precursors and leading to photosynthetic pigment degradation. The chlorophyll loss further compromises membrane structural integrity, ultimately impairing PSII (for example, a decrease in Fv/Fm values). 

Comments 28:  The Discussion could benefit from including the study limitations, future research suggestions, implications for cultivation, breeding, or stress management.

Response 28: We fully agree with the reviewers' valuable suggestions and express our gratitude. We have added the following to the discussion section: In this study, we systematically investigated the effects of high-temperature stress on the photosynthetic characteristics, chlorophyll synthesis, and tissue structure of ginseng leaves. However, there are still some aspects requiring further research. Firstly, the key structural parameters, such as stomatal density, thylakoid membrane thickness, and number of stacked basal granules, need to be quantitatively analyzed. Secondly, the activities of other key enzymes in the chlorophyll biosynthesis pathway, except ALAD and PBGD, were not detected. Thirdly, the expression regulation network of photosynthesis-related genes in response to high temperature has not been fully elucidated at the molecular level. In the future, we intend to further deepen the quantitative characterization of the organization structure, improve the chlorophyllase detection system in the chlorophyll synthesis pathway, and focus on the molecular mechanism of the related gene expression. On this basis, the heat resistance of ginseng may be improved by combining the practice of heat-resistant germplasm screening and genetic improvement, providing support for the stress-resistant breeding of ginseng and the sustainable development of the industry.

Comments 29: 4.1. Strong claims (“physiological vitality diminishes”) need more quantitative support. Causality between RWC decline and chloroplast damage is speculative unless proven. I suggest the authors to link their own data on RWC and stomatal. The explanation for stomatal closure prioritizes dehydration, but high temperature can also increase vapor pressure deficit, this nuance is missing.

Response 29: We fully endorse the reviewers' valuable feedback and extend our sincere gratitude. We have revised the overly assertive statements in the discussion section to enhance the rigor of our conclusions. Furthermore, we have conducted a correlation analysis between RWC and stomatal parameters, and restructured the relevant research content accordingly.

Comments 30: 4.2. The paragraph is overly long, repetitive, and contains internal contradictions. It claims both accelerated chlorophyll biosynthesis and inhibited biosynthesis. Mechanisms such as “cutting off the normal supply of metabolic substrates” are not measured experimentally. The explanation of precursor accumulation and blockages is speculative without measuring Mg-chelatase, CHLH, CHLD, POR, etc. I suggest the authors to focus on what the study measured: decreased precursors, decreased ALAD/PBGD and decreased pigment content. And to discuss potential upstream/downstream mechanisms only as hypotheses.

Response 30: We fully endorse the reviewers' valuable feedback and extend our gratitude. The relevant sections have been revised, and the discussion has been adjusted accordingly. For details, please refer to Section 4.2 of the manuscript.

Comments 31:  4.3. Over-emphasizes literature rather than connecting to study own data. Logical interpretation of Ci and Gs is correct but oversimplified (mesophyll conductance and biochemical limitations are not discussed). The authors should clarify that Ci rise does not prove  non-stomatal limitation, it is consistent with it. Attribution of Fo increase solely to RC closure is too simplistic; antenna uncoupling also possible. The text does not address whether NPQ decrease implies impaired heat dissipation.

Response 31: We fully endorse the reviewers' valuable feedback and extend our gratitude. The relevant sections have been revised and expanded, as detailed in Section 4.3 of the manuscript.

Comments 32:  4.4.  The authors used very strong wording (“severely damaged,” “ruptures,” “irreversible”). There is no quantitative metrics from TEM (e.g., grana number, thylakoid spacing). Mitochondria are mentioned even though not measured. Discussion of stomata focuses on size but ignores density. Suggest to add a note: stomatal density not measured, could affect conclusions. suggest to add nuance: ultrastructural changes may reflect combined heat and water stress.

Response 32: We fully endorse the reviewers' valuable feedback and extend our gratitude. All overly absolute qualitative descriptions in the results section (e.g., "severely impaired,"  "irreversible") have been revised to objective language. We completely agree with your point. Since the initial design of this study focused primarily on observing changes in chloroplast morphology, we have clearly stated this in the 'Materials and Methods' section. During the experimental design phase, we did not include stomatal density as a detection indicator, mainly because stomatal length and width can directly reflect changes in turgor pressure of guard cells and stomatal movement status under high-temperature conditions. This study elucidates how high temperatures regulate stomatal opening/closing to mediate gas exchange and water loss in leaves, which constitutes the core focus. We concur with your observation that stomatal density measurements could enhance the research framework for understanding temperature effects on stomata. Consequently, we acknowledge this limitation in the discussion section, with future studies aiming for quantitative analysis. After verification, mitochondrial TEM results were excluded from the text, and we have removed speculative discussions related to this aspect. In Section 4.4, it is supplemented that ultrastructural changes may reflect the combined effects of thermal stress and water stress. The specific content is as follows: The results indicate that high-temperature stress forced RWC to decrease, leading to cellular dehydration and disrupting the integrity of chloroplast structure, causing damage to the photosynthetic machinery, and severely affecting the plant's photosynthesis.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The submitted manuscript “Research on the response mechanism of the photosynthetic system of Panax ginseng leaves to high-temperature stress” is focused on the effects of three high temperature treatments of Panax ginseng plants for 12, 24, 36 and 48 h and the alterations in photosynthetic performance. In general, the results could represent interest, however, there are serious flaws that need to be addressed before the manuscript to be considered suitable for publication.

• English language needs serious improvement in respect to staling, grammar, typing errors. When describing the results is better to use past tense.
• Reformulation for better clarity is required: line 61-63, 70-75,
• Line 65 - “photosynthesis mechanism” to be corrected to “photosynthetic process”
• Line 66 – “Chlorophyll biosynthesis is a series of continuous enzymatic reaction processes” – reformulate
• Line 84-86 – the effect of high on photosynthetic performance is very intensively investigated, both on leaves and thylakoid membranes - reformulate
• Line 92 – “flower pots” – just say “pots”
• Section Materials and Methods – everywhere when the devise is used for investigation, please, provide the model, organization, town and country.
• Line 98-99 – repetition of the information from subsection 2.1.
• Line 106 – “healthy plants without pests and diseases” – does it mean that some plants were infected under the control conditions?
• Line 113 – “backed” is not appropriate. Dry weight was determined after keeping the leaves in an oven for at least 24 h or more, while reaching the dry weight. In addition, the given formulae for calculation of RWC is not correct. It is the formulae for determination of leaf water content, which is different. Leaf water content is the total amount of water in a leaf, while relative water content (RWC) is a percentage that compares the leaf's current water content to its maximum possible water content when fully hydrated. In the context of research presented it is more appropriate to present RWC, which is the measure of hydrogen state of the leaf and normalizes the measurement for the leaf’s size and structure. In the whole text the two parameters are used with presumption that it is one and the same, but they are not equal.
• Subsection 2.4. – Give the full name and model of the PAM fluorometer as well as the formulas for calculation of Fv/Fm and NPQ and a reference.
• For subsections 2.5 and 2.6 no references are provided. They should be given.
• Line 135 – “basic acetone” – what does it mean?
• Line 136 – “volume was fixed at 5 mL.” – paraphrase.
• Line 138 – “calculate the contents of Proto IX, Mg-proto IX and Pch I.” – How the calculation was performed? Provide a reference.
• Subsection 2.7 – For determination of pigment content a reference is given, 28, Zhang, Z.; Chen, Z. Experimental techniques in plant physiology. Jilin University Press: Jilin, 2012. In this case is more appropriate to cite the original manuscript where are provided the solvents, coefficients for the respective pigment and the formulae for calculation. I hardly believe that the scientists from Jilin University established the procedure, coefficients and formulas. Most probably they used the manuscript of Lichtenthaler 1987, Methods of Enzymology.
• Line 155, 168 – “100%, and 100%” probably the second 100% is extra.
• Line 163 and 176 – light or electron microscope?
• Line 197 – it is not “9.88%” but “9%”
• Line 217-219 – reformulate for clarity
• Line 234-237 – Reformulate
• In table 2 are presented values only of control plants while the parameters of “40/28℃ (high-temperature stress for 48 h).” are missing.
• It is obvious (Figure 5) that the greatest decline in pigment content, as induced by high temperature, is observed in carotenoid content. Throughout the text, results and discussion, the attention is focused mainly on chlorophyll content and the decline in carotenoids is neglected. Carotenoids are important component of photosynthetic apparatus performing multiple functions. To be corrected accordingly.
• To correct appropriately the figure 6 legend
• When chloroplasts are concerned, we have thylakoid membranes, which are stroma exposed, not appressed, and appressed that form granna. So, the expression “thylakoid sheets” is not correct. “granum layer” is also not correct – it is granna. Line 339-346 and legend of figure 7 to be corrected accordingly. In figure 6 and 7 the bars indicating the magnification should be included in every panel, not given in the figure legend. In figure 7 d, the quality is so low that single stroma exposed thylakoid membranes are hardly to be observed.
• Line 445-448 – To be reformulated and “tion” in line 446 to be removed.
• Line 465 – “eaves” – to be removed.
• Line 466-467, 482-484 – to be reformulated.
• Line 513 – “also suffered irreversible damage” – this statement is not correct as no results were presented about recovery from the high-temperature stress.

As the manuscript suffers serious flaws my recommendation is to reject the manuscript in order the authors to have enough time for corrections and to be resubmitted again.

Author Response

Comments 1: English language needs serious improvement in respect to staling, grammar, typing errors. When describing the results is better to use past tense.

Response 1: We fully endorse the reviewers' valuable feedback and extend our gratitude. The manuscript has been refined using the journal's language editing service to ensure more precise and clear presentation of our research findings.

Comments 2: Reformulation for better clarity is required: line 61-63, 70-75.

Response 2: We fully endorse the reviewers' valuable feedback and extend our gratitude. We have revised the relevant section to clarify the research content. Additionally, in response to the deficiencies in the preface identified by Reviewers 1 and 2, we have made corresponding revisions to the preface.

Comments 3: Line 65 - “photosynthesis mechanism” to be corrected to “photosynthetic process”.

Response 3: We fully endorse the reviewer's valuable suggestions and extend our gratitude. We have revised “photosynthesis mechanism” to “photosynthetic process”. Additionally, as reviewers 1 and 2 provided feedback on the preface, we have made corresponding revisions to the preface content, as detailed in the manuscript's preface section.

Comments 4: Line 66 – “Chlorophyll biosynthesis is a series of continuous enzymatic reaction processes” – reformulate

Response 4: We fully agree with the reviewer's valuable comments and thank you. We have made the following revisions to this section: Chlorophyll, as the core pigment for photosynthesis in higher plants, has a complex biosynthetic process involving multiple enzymes. 

Comments 5: Line 84-86 – the effect of high on photosynthetic performance is very intensively investigated, both on leaves and thylakoid membranes - reformulate

Response 5: We fully endorse the reviewers' valuable feedback and extend our gratitude. We have revised the relevant sections accordingly. Additionally, as reviewers 1 and 2 provided suggestions on the preface, we have also updated the preface content. For details, please refer to the preface section of the manuscript.

Comments 6: Line 92 – “flower pots” – just say “pots”

Response 6: We fully endorse the reviewers' valuable feedback and extend our sincere thanks. In line 92 of the manuscript, we have revised “flower pots” to “pots”.

Comments 7: Section Materials and Methods – everywhere when the devise is used for investigation, please, provide the model, organization, town and country.

Response 7: We fully endorse the reviewers' valuable suggestions and extend our gratitude. All instruments referenced in the materials and methods section have been properly labeled with their model numbers, affiliated institutions, locations, and countries. The revised content is detailed in the manuscript.

Comments 8: Line 98-99 – repetition of the information from subsection 2.1. 

Response 8: We fully agree with the reviewers' valuable suggestions and thank them. We have removed duplicate content from the manuscript.

Comments 9: Line 106 – “healthy plants without pests and diseases” – does it mean that some plants were infected under the control conditions?

Response 9: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. All experimental materials in the trial group (including the control group) were selected from healthy, uniformly grown plants free from pest or disease infestations. Recognizing the original wording's inadequacy, we have revised the relevant section by replacing “without pests and diseases” with “healthy and uniformly grown plants” to more accurately reflect our material selection criteria.

Comments 10: Line 113 – “backed” is not appropriate. Dry weight was determined after keeping the leaves in an oven for at least 24 h or more, while reaching the dry weight. In addition, the given formulae for calculation of RWC is not correct. It is the formulae for determination of leaf water content, which is different. Leaf water content is the total amount of water in a leaf, while relative water content (RWC) is a percentage that compares the leaf's current water content to its maximum possible water content when fully hydrated. In the context of research presented it is more appropriate to present RWC, which is the measure of hydrogen state of the leaf and normalizes the measurement for the leaf’s size and structure. In the whole text the two parameters are used with presumption that it is one and the same, but they are not equal.

Response 10: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. In line 113, we have revised “backed” to “subjected to baking”. Furthermore, we have clarified the conceptual distinction between the two terms, corrected “Leaf relative water content” to “relative water content (RWC)”, and updated the relevant formulas accordingly.

Comments 11: Subsection 2.4. – Give the full name and model of the PAM fluorometer as well as the formulas for calculation of Fv/Fm and NPQ and a reference. 

Response 11: We fully agree with the reviewers' valuable comments and express our gratitude. We have supplemented the full name and model of the PAM fluorometer, as well as the calculation formulas for Fv/Fm and NPQ, and included references. See references 17,18.

Comments 12: For subsections 2.5 and 2.6 no references are provided. They should be given.

Response 12: We fully endorse the reviewers' valuable comments and express our gratitude. 2.5 Determination of Key Enzyme Activity Indicators for Chlorophyll Precursors: The activities of ALAD and PBGD enzymes were measured using kit-based assays. We have supplemented the methodology in the manuscript section 2.5, therefore no references were cited. 2.6 Selected References: Refer to references 17 and 18.

Comments 13: Line 135 – “basic acetone” – what does it mean?

Response 13: We fully endorse the reviewers 'valuable feedback and extend our sincere gratitude. We apologize for the language discrepancy regarding “alkaline acetone” and have made corresponding revisions in the manuscript.

Comments 14: Line 136 – “volume was fixed at 5 mL.” – paraphrase.

Response 14: We fully endorse the reviewers' valuable suggestions and extend our gratitude. We have revised the phrasing from “volume was fixed at 5 mL” to “volume was adjusted to 5 mL”.

Comments 15: Line 138 – “calculate the contents of Proto IX, Mg-proto IX and Pch I.” – How the calculation was performed? Provide a reference.

Response 15: We fully endorse the reviewers' valuable suggestions and extend our gratitude. We have supplemented the manuscript with references for calculating the contents of Proto IX, Mg-proto IX, and Pchl, as detailed in References 20 and 21.

Comments 16: Subsection 2.7 – For determination of pigment content a reference is given, 28, Zhang, Z.; Chen, Z. Experimental techniques in plant physiology. Jilin University Press: Jilin, 2012. In this case is more appropriate to cite the original manuscript where are provided the solvents, coefficients for the respective pigment and the formulae for calculation. I hardly believe that the scientists from Jilin University established the procedure, coefficients and formulas. Most probably they used the manuscript of Lichtenthaler 1987, Methods of Enzymology. 

Response 16: We fully endorse the reviewers' valuable feedback and extend our gratitude. The literature has been revised accordingly. Additionally, as Reference 1 raised issues with the references, we have updated them. The number of references has been reduced from 28 to 22, as shown in Reference 22.

Comments 17: Line 155, 168 – “100%, and 100%” probably the second 100% is extra.

Response 17: We fully agree with the reviewer's valuable comments and thank you. We have deleted the second 100%.

Comments 18: Line 163 and 176 – light or electron microscope?

Response 18: We fully agree with the reviewers' valuable suggestions and express our gratitude. To observe the stomatal structure characteristics of ginseng leaves, we selected scanning electron microscopy (SEM).

Comments 19: Line 197 – it is not “9.88%” but “9%”

Response 19: We fully agree with the reviewer's valuable suggestions and express our gratitude. We have changed column 197 from “9.88%” to “9%”.

Comments 20: Line 217-219 – reformulate for clarity 

Response 20: We fully endorse the reviewers' valuable feedback and express our gratitude. The relevant content has been rephrased. As reviewer 2 suggested, we moved this section to the discussion section and made corresponding revisions. For details, please refer to the manuscript.

Comments 21: Line 234-237 – Reformulate

Response 21: We fully endorse the reviewers' valuable feedback and express our gratitude. The relevant content has been rephrased. As reviewer 2 suggested, we moved this section to the discussion section and made corresponding revisions. For details, please refer to the manuscript.

Comments 22: In table 2 are presented values only of control plants while the parameters of “40/28℃ (high-temperature stress for 48 h).” are missing.

Response 22: We fully endorse the reviewers' valuable feedback and extend our gratitude. The relevant sections have been updated, with specific details available in the manuscript.

Comments 23: It is obvious (Figure 5) that the greatest decline in pigment content, as induced by high temperature, is observed in carotenoid content. Throughout the text, results and discussion, the attention is focused mainly on chlorophyll content and the decline in carotenoids is neglected. Carotenoids are important component of photosynthetic apparatus performing multiple functions. To be corrected accordingly. 

Response 23: We fully agree with the reviewer's valuable comments and express our gratitude. We have supplemented this section with the following content:  It is particularly noteworthy that the content of carotenoids decreases most significantly under high-temperature conditions. The carotenoids were degraded by heat, and the chlorophyll content was reduced.

Comments 24: To correct appropriately the figure 6 legend

Response 24: We fully endorse the reviewers' valuable feedback and extend our gratitude. The legend for Figure6 has been revised as follows: Figure 6. The effect of high temperature stress on gaseous parameters of ginseng stomata. (a) Stomatal parameter of ginseng leaves at 25/16 ℃ (20 μm, ×2.00k (2000×)); (b) Stomatal parameters of ginseng leaves at 40/28 ℃ high temperature stress for 48 h  (20 μm, ×2.00k (2000×)).

Comments 25: When chloroplasts are concerned, we have thylakoid membranes, which are stroma exposed, not appressed, and appressed that form granna. So, the expression “thylakoid sheets” is not correct. “granum layer” is also not correct – it is granna. Line 339-346 and legend of figure 7 to be corrected accordingly. In figure 6 and 7 the bars indicating the magnification should be included in every panel, not given in the figure legend. In figure 7 d, the quality is so low that single stroma exposed thylakoid membranes are hardly to be observed.

Response 25: We fully endorse the reviewers 'valuable feedback and extend our gratitude. We have corrected the inaccurate term' thylakoid lamella 'and revised the relevant section. The term' basal granule layer 'has been updated to' basal granule'. The magnification scale bar in Figure 6 is located at the lower left corner of the image, where ×2.00 k (2000×); in Figure 7, the magnification scale bar in Figure 6 is also located at the lower left corner of the image, where ×1.0 k (1000×),×4.0 k (4000×). The image quality of Figure 7 has been modified, and the column in Figure 7 has also been modified. The specific modifications are as follows: Figure 7. The effect of high temperature stress on the ultrastructure of ginseng chloroplasts. a,b. Ultrastructural of chloroplasts in ginseng leaves at 25/16 ℃ (CK, 10 um); c,d. Ultrastructural of chloroplasts in ginseng leaves at 40/28 ℃ (high temperature stress for 48 h, 20 um). Note: The magnification of images a and c is ×1.0 k (1000×), and that of images b and d is ×4.0 k (4000×). Chl: chloroplast; GL: Grana; OG: Osmophilic granules.

Comments 26: Line 445-448 – To be reformulated and “tion” in line 446 to be removed.

Response 26: We fully agree with the reviewer's valuable feedback and express our gratitude. We have rephrased this section and removed the word "tion" in line 446. For details, please refer to the manuscript.

Comments 27: Line 465 – “eaves” – to be removed.

Response 27: We fully agree with the reviewer's valuable comments and thank you. We have removed "eaves" from line 446.

Comments 28: Line 466-467, 482-484 – to be reformulated.

Response 28: We fully endorse the reviewers' valuable feedback and extend our gratitude. The relevant section has been rephrased, with specific revisions available in the manuscript.

Comments 29: Line 513 – “also suffered irreversible damage” – this statement is not correct as no results were presented about recovery from the high-temperature stress.

Response 29: We fully agree with the reviewer's valuable feedback and thank you. First, we apologize. We have recognized the error in the statement and have corrected it.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I have carefully reviewed your revised manuscript and am pleased to note that you have responded thoroughly to my earlier suggestions. The changes you have made have improved the quality and clarity of the work, and I believe it is ready for publication in Horticulturae.

Author Response

Comments 1:

Dear Authors,

I have carefully reviewed your revised manuscript and am pleased to note that you have responded thoroughly to my earlier suggestions. The changes you have made have improved the quality and clarity of the work, and I believe it is ready for publication in Horticulturae.

Reponse 1: 

Dear Reviewer,

We sincerely appreciate your thorough review of our revised manuscript and your endorsement of the work. We are pleased to hear that the quality of the revised manuscript has been enhanced and is now ready for publication in Horticulturae. We are deeply honored and gratified. We sincerely appreciate your valuable and instructive suggestions throughout the entire peer review process. It is your professional guidance and rigorous review that have significantly enhanced the rigor and clarity of our research. We have taken every suggestion you provided very seriously, which has been a valuable learning and improvement process for us. We would like to express our sincere gratitude for your time and effort, and thank the journal editorial board for providing us with valuable opportunities for communication and improvement. We look forward to presenting our research findings in Horticulturae. Wishing you smooth progress and good health.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version of the submitted manuscript was significantly improved. However, most of the critical comments in the first round of reviewing were not addressed, nevertheless that the authors state in their response that all recommendations were taken into account, which is not the case.

• L 96 – “illuminance” to be changed to “illumination”
• L 114 – When the dry weight is determined, leaves are not “backed” in oven but “dried
• L 115 – “to keel bacteria” does not have place here. Leaves are in oven to remove the water.
• L 122 – “Britain” to be replaced by “UK”
• L 130 – “USA” to be removed. “Czech Republic” – it is not part of USA but independent country in Europe!
• L 132-134 – To be corrected. The given formulas to be placed properly. The given formulae for NPQ is not correct. In addition, for F0 (use “o” but not “0”), Fm and Fv/Fm – o, v and m should be in subscript, here and everywhere in the text.
• L 142, 144, 156 – “uL” it seems it is a typing error; it should be “µL”.
• L 151, 152, 165, 166 – instead OD it is more scientific to use “absorbance”
• L 183 – “Shanghai, Chian)” – again a typing error, probably it has to be “China”
• Everywhere in the text, Chl a and Chl b – a and b should be in Italic.
• Table 1 – The caption of the figure and the values are the same as in the previous version. It is again “water content”, nevertheless that in the authors’ response is stated that is corrected to “RWC”
• Figure 1 legend – “photosynthesis” is not correctly used. In the figure are presented “gas exchange parameters” that are related to photosynthesis. The quality of the figure is very low! To be improved. The quality of figure 2 and 5 is also very low. To be improved.
• L 306 – “chlorophyll” to be corrected to “pigment”
• The stronger effect of high temperature on carotenoid content is only mentioned in the description of results and in Discussion section without any interpretation of their functions and consequences for the photosynthetic performance.
• L 330 – “Figure 6. The effect of high-temperature stress on gaseous parameters” – it is not correct. In the figure are presented the images of stomata, control and at high temperature. To be corrected. In the panels are not included the lines, indicating the magnifications, as was stated in the authors’ response. The lines are also missing in figure 7. They should be in the panels, not in the legend!
• L 379 – “Effects of high-temperature stress on water metabolism and stoma in ginseng leaves” – “metabolism” is not used correctly. Authors are presenting only RWC and not mechanism.
• L 498 – “stacked basal granules” – not correct.
• L 633 – “Mahonia bodinieri” should be in italic.

 

My general conclusion is that the manuscript still is not suitable for acceptance to be published.

Comments on the Quality of English Language

The English language is fine

Author Response

Dear Reviewer: 

We have submitted our point-by-point responses to your comments in a formal document. Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript was significantly improved.

It can be accepted for publication, if the Editor finds it reasonable. 

Author Response

Comments 1: The manuscript was significantly improved. It can be accepted for publication, if the Editor finds it reasonable. 

Reponse 1: Dear Reviewer, We sincerely appreciate the time and expertise you dedicated to reviewing our manuscript. Your valuable feedback has been instrumental in improving the quality of our manuscript. We look forward to presenting our research findings in Horticulturae. Wishing you smooth progress and good health.